Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Variations and reduction of plastome are associated with the evolution of parasitism in Convolvulaceae.

Parasitic lifestyle can often relax the constraint on the plastome, leading to gene pseudogenization and loss, and resulting in diverse genomic structures and rampant genome degradation. Although several plastomes of parasitic Cuscuta have  been reported, the evolution of parasitism in the family Convolvulaceae which is linked to structural variations and reduction of plastome has not been well investigated. In this study, we assembled and collected 40 plastid genomes belonging to 23 species representing four subgenera of Cuscuta and ten species of autotrophic Convolvulaceae. Our findings revealed nine types of structural variations and six types of inverted repeat (IR) boundary variations in the plastome of Convolvulaceae spp. These structural variations were associated with the shift of parasitic lifestyle, and IR boundary shift, as well as the abundance of long repeats. Overall, the degradation of Cuscuta plastome proceeded gradually, with one clade exhibiting an accelerated degradation rate. We observed five stages of gene loss in Cuscuta, including NAD(P)H complex → PEP complex → Photosynthesis-related → Ribosomal protein subunits → ATP synthase complex. Based on our results, we speculated that the shift of parasitic lifestyle in early divergent time promoted relaxed selection on plastomes, leading to the accumulation of microvariations, which ultimately resulted in the plastome reduction. This study provides new evidence towards a better understanding of plastomic evolution, variation, and reduction in the genus Cuscuta.

Cuscuta chinensis flavonoids alleviate ovarian damage in offspring female mice induced by BPA exposure during pregnancy by regulating the central carbon metabolism pathway.

Pregnancy is a sensitive window period for bisphenol A (BPA) exposure. BPA can pass through the placenta and cause reproductive damage in offspring female mice. Even BPA that is not metabolized during lactation can be passed through milk. Cuscuta chinensis flavonoids (CCFs) can alleviate reproductive damage caused by BPA, but the mechanism of action is unclear. To investigate the potential mitigating impact of CCFs on ovarian damage resulting from BPA exposure during pregnancy, we administered BPA and CCFs to pregnant mice during the gestational period spanning from 0.5 to 17.5 days. Aseptic collection of serum and ovaries from female mice was conducted on postnatal day 21 (PND21). Serum hormone levels and tissue receptor levels were quantified utilizing ELISA and PCR, while ovaries underwent sequencing and analysis through transcriptomics and metabolomics techniques. Additionally, the assessment of ovarian oxidative stress levels was carried out as part of the comprehensive analysis. The results showed that CCFs administration mitigated the adverse effects induced by BPA exposure on ovarian index, hormone levels, receptor expression, and mRNA expression levels in female offspring mice. The joint analysis of transcriptome and metabolome revealed 48 enriched pathways in positive ion mode and 44 enriched pathways in negative ion mode. Among them, the central carbon metabolism pathway is significantly regulated by BPA and CCFs. The screened sequencing results were verified through qPCR and biochemical kits. In this study, CCFs may participate in the central carbon metabolism pathway by reducing the expression of Kit proto-oncogene (Kit), hexokinase 1 gene (Hk1) and pyruvate kinase M (Pkm) mRNA and increasing the expression of h-ras proto-oncogene (Hras), sirtuin 3 (Sirt3), sirtuin 6 (Sirt6) and TP53 induced glycolysis regulatory phosphatase gene (Tigar) mRNA, thereby resisting the effects of BPA on the body. At the same time, the metabolic levels of D-Fructose 1,6-bisphosphate and L-Asparagine tend to be stable. Moreover, CCFs demonstrated a capacity to diminish the BPA-induced escalation in reactive oxygen species (ROS) and malondialdehyde (MDA). Simultaneously, it exhibited the ability to elevate levels of glutathione (GSH) and catalase (CAT), thereby effectively preventing peroxidation. In summary, CCFs alleviate BPA-induced ovarian damage in offspring female mice by regulating the central carbon metabolism pathway. This study will improve the information on BPA reproductive damage antagonist drugs and provide a theoretical basis for protecting animal reproductive health.

Cuscuta chinensis Lam. Flavonoids (CCLF) alleviate the symptoms of sepsis-associated encephalopathy via PI3K/Nrf2 pathway.

Sepsis-associated encephalopathy (SAE) frequently encounters patients who are in intensive care units and ∼70% of patients with severe systemic infection. However, due to the unclear pathological mechanisms of SAE, the desease-modifying drug is still lack. Here, we aimed to explore whether the flavonoid components extracted from CCL (CCLF) seeds possess protective effects on SAE animals, and systematically evaluate the transcriptomic alteration (in the hippocampus) after CCLF treatment on SAE animals employing RNA sequencing. We observed that CCLF improved the brain's learning and memory abilities and the structural integrity of BBB using cecal ligation and puncture (CLP)-induced SAE animal models, evaluated by behavioral test and tissue examination of animals respectively. RNA sequencing results showed that CCLF treatment reverses SAE-induced transcriptomic alteration in the hippocampus. Moreover, CCLF also dramatically relieved inflammatory (such as TNF-α, IL-2, and IL-6) and oxidative (MDA and SOD activity) stresses, and inhibited SAE-induced neuron apoptosis in brain tissues. More importantly, CCLF restored the PI3K/AKT signaling pathway and then induced the Nrf2 nuclear translocation to drive HO-1 expression both in vitro and in vivo. LY294002, an inhibitor of PI3K, obviously blocked CCLF's functions on anti-apoptosis, anti-inflammation, and anti-oxidation in vivo, demonstrating that CCLF achieves its bioactivities in a PI3K/AKT signaling dependent manner. Altogether, CCLF exhibits remarkable neuro-protective function and may be a promising candidate for further clinical trials for SAE treatment.

Rising from the shadows: Selective foraging in model shoot parasitic plants.

Despite being sessile, plants nonetheless forage for resources by modulating their growth. Adaptative foraging in response to changes in resource availability and presence of neighbours has strong implications for performance and fitness. It is an even more pressing issue for parasitic plants, which draw resources directly from other plants. Indeed, parasitic plants were demonstrated over the years to direct their growth towards preferred hosts and invest resources in parasitism relative to host quality. In contrast to root parasites that rely mostly on chemical cues, some shoot parasites seem to profit from the ability to integrate different types of abiotic and biotic cues. While significant progress in this field has been made recently, there are still many open questions regarding the molecular perception and the integration of diverse signalling pathways under different ecological contexts. Addressing how different cues are integrated in parasitic plants will be important when unravelling variations in plant interaction pathways, and essential to predict the spread of parasites in natural and agricultural environments. In this review, we discuss this with a focus on Cuscuta species as an emerging parasitic model, and provide research perspectives based on the recent advances in the topic and plant-plant interactions in general.

Cuscuta campestris fine-tunes gene expression during haustoriogenesis as an adaptation to different hosts.

The Cuscuta genus comprises obligate parasitic plants that have an unusually wide host range. Whether Cuscuta uses different infection strategies for different hosts or whether the infection strategy is mechanistically and enzymatically conserved remains unknown. To address this, we investigated molecular events during the interaction between field dodder (Cuscuta campestris) and two host species of the Solanum genus that are known to react differently to parasitic infection. We found that host gene induction, particularly of cell wall fortifying genes, coincided with a differential induction of genes for cell wall degradation in the parasite in the cultivated tomato (Solanum lycopersicum) but not in a wild relative (Solanum pennellii). This indicates that the parasite can adjust its gene expression in response to its host. This idea was supported by the increased expression of C. campestris genes encoding an endo-ß-1,4-mannanase in response to exposure of the parasite to purified mono- and polysaccharides in a host-independent infection system. Our results suggest multiple key roles of the host cell wall in determining the outcome of an infection attempt.

A roadmap of haustorium morphogenesis in parasitic plants.

Parasitic plants invade their host through their invasive organ, the haustorium. This organ connects to the vasculature of the host roots and hijacks water and nutrients. Although parasitism has evolved independently in plants, haustoria formation follows a similar mechanism throughout different plant species, highlighting the developmental plasticity of plant tissues. Here, we compare three types of haustoria formed by the root and shoot in the plant parasites Striga and Cuscuta. We discuss mechanisms underlying the interactions with their hosts and how different approaches have contributed to major understanding of haustoria formation and host invasion. We also illustrate the role of auxin and cytokinin in controlling this process.

Complete chloroplast genomes of five Cuscuta species and their evolutionary significance in the Cuscuta genus.

BACKGROUND: Cuscuta, a parasitic plant species in the Convolvulaceae family, grows in many countries and regions. However, the relationship between some species is still unclear. Therefore, more studies are needed to assess the variation of the chloroplast (cp) genome in Cuscuta species and their relationship with subgenera or sections, thus, providing important information on the evolution of Cuscuta species. RESULTS: In the present study, we identified the whole cp genomes of C. epithymum, C. europaea, C. gronovii, C. chinensis and C. japonica, and then constructed a phylogenetic tree of 23 Cuscuta species based on the complete genome sequences and protein-coding genes. The complete cp genome sequences of C. epithymum and C. europaea were 96,292 and 97,661 bp long, respectively, and lacked an inverted repeat region. Most cp genomes of Cuscuta spp. have tetragonal and circular structures except for C. epithymum, C. europaea, C. pedicellata and C. approximata. Based on the number of genes and the structure of cp genome and the patterns of gene reduction, we found that C. epithymum and C. europaea belonged to subgenus Cuscuta. Most of the cp genomes of the 23 Cuscuta species had single nucleotide repeats of A and T. The inverted repeat region boundaries among species were similar in the same subgenera. Several cp genes were lost. In addition, the numbers and types of the lost genes in the same subgenus were similar. Most of the lost genes were related to photosynthesis (ndh, rpo, psa, psb, pet, and rbcL), which could have gradually caused the plants to lose the ability to photosynthesize. CONCLUSION: Our results enrich the data on cp. genomes of genus Cuscuta. This study provides new insights into understanding the phylogenetic relationships and variations in the cp genome of Cuscuta species.

Inflorescences of Cuscuta (Convolvulaceae): Diversity, evolution and relationships with breeding systems and fruit dehiscence modes.

Cuscuta (dodder) includes ca. 200 species of plant obligate stem parasites with enormous ecological and economical significance. Inflorescences have been historically used in Cuscuta for species descriptions and identification keys, but no comprehensive study exists to date. The main objectives of this study were to survey the diversity and evolution of inflorescences and to uncover their possible form-function relationships. The inflorescence architecture of 132 Cuscuta taxa was analysed using herbarium specimens and eight species were grown to study their inflorescence development. Inflorescence traits were mapped into a genus phylogeny obtained from a combined analysis of nuclear ITS and plastid trnL-F sequences. To test the hypothesis that inflorescence architecture is connected to sexual reproduction, correlations between inflorescence traits (using Principal Components), sexual reproductive traits (pollen/ovule ratios, corolla length and diameter), fruit charaters (fruit length and width), and the modes of dehiscence were analyzed. Based on their development, three major types of inflorescences were observed: "Cuscuta type", a simple, monochasial scorpioid cyme; "Monogynella type", a compound monochasial scorpioid cymes with the longest primary axes having prolonged vegetative growth and giving the appearance of thyrses; and "Grammica type", a compound monochasial scorpiod cymes with up to five orders of axes. Maximum likelihood analyses suggested Monogynella as the ancestral type, while Cuscuta and Grammica were derived. Overall, the total length of axes exhibited a reduction trend throughout the genus evolution, but it was not correlated with the pedicels length. Inflorescences with similar architectures may exhibit contrasting pollen-ovule ratios. Positive significant correlations were noted between the size of the flower traits and pollen-ovule ratios. Several modes of dehiscence had statistically significant different total axes lengths, suggesting that the infructescence architecture is connected to the modes of dehiscence in Cuscuta and therefore seed dispersal.

Disruption of the standard kinetochore in holocentric Cuscuta species.

The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

Phytochemical Investigation of Cuscuta campestris Yunck. Stem Extract and Evaluation of Its Bioherbicidal Effect on Amaranthus retroflexus L. and Portulaca oleracea L.

This study focused on characterizing chemically and evaluating in vitro allelopathic and bioherbicidal potential of secondary metabolites extracted from the stem of Cuscuta campestris in seed germination, early seedling growth and early plant growth of Amaranthus retroflexus and Portulaca oleracea. The combined effects of stem extract and a reduced dose of herbicide metribuzin were also examined. Plant extract contained 17 phenolic compounds and the most abundant phenols were flavonoids: quercetin, (+)-catechin, daidzin, luteolin, and rutin. The seeds of P. oleracea were less sensitive than the seeds of A. retroflexus. The seed bioassay confirmed the inhibitory effect of stem extract on germination and early growth of both weed seedlings at concentrations of 0.75 % and 1 %, and a minor inhibitory effect in the plant bioassay. On the other hand, a synergy of C. campestris stem extract and metribuzin was revealed, as their combination achieved better results in the control of both weed species. Based on obtained data C. campestris stem extract could be a potential source of natural-based weed control molecules.

Cuscuta reflexa Possess Potent Inhibitory Activity Against Human Malaria Parasite: An In Vitro and In Vivo Study.

Drug resistance to practically all antimalarial drugs in use necessitate the development of new chemotherapeutics against malaria. In this aspect, traditionally used plants with folklore reputation are the pillar for drug discovery. Cuscuta reflexa being traditionally used in the treatment of malaria in Odisha, India we aimed to experimentally validate its antimalarial potential. Different solvent extracts of C. reflexa or column fractions from a promising solvent extract were evaluated for in vitro anti-plasmodial activity against Plasmodium falciparum strain Pf3D7. Potent fractions were further evaluated for inhibition of parasite growth against different drug resistant strains. Safety of these fractions was determined by in vitro cyto-toxicity, and therapeutic effectiveness was evaluated by suppression of parasitemia and improvement in survival of experimental mice. Besides, their immunomodulatory effect was investigated in Pf-antigen stimulated RAW cells. GCMS fingerprints of active fractions was determined. Column separation of methanol extract which showed the highest in vitro antiplasmodial activity (IC50 = 14.48 µg/ml) resulted in eleven fractions, three of which (F2, F3, and F4) had anti-plasmodial IC50 ranging from ≤ 10 to 2.2 µg/ml against various P. falciparum strains with no demonstration of in vitro cytotoxicity. F4 displayed the highest in vivo parasite suppression, and had a mean survival time similar to artesunate (19.3 vs. 20.6 days). These fractions significantly modulated expression of inflammatory cytokines in Pf-antigen stimulated RAW cells. The findings of the study confirm the antimalarial potential of C. reflexa. Exploration of phyto-molecules in GCMS fingerprints of active fractions is warranted for possible identification of lead anti-malarial phyto-drugs.

Cuscuta chinensis flavonoids reducing oxidative stress of the improve sperm damage in bisphenol A exposed mice offspring.

Bisphenol A (BPA) is a common environmental endocrine disruptor, and overexposure is a threat to male reproduction. Although studies have confirmed that BPA exposure causes a decrease in sperm quality in offspring, the dosage used, and the underlying mechanism is not clear. The purpose of this study is to investigate whether Cuscuta chinensis flavonoids (CCFs) can antagonize or alleviate BPA-induced reproductive injury by analyzing the processes associated with BPA's impairment of sperm quality. BPA and 40 mg/kg bw/day of CCFs were administered to the dams at gestation day (GD) 0.5-17.5. Testicles and serum of male mice are collected on postnatal day 56 (PND56), and spermatozoa are collected to detect relevant indicators. Our results showed that compared with the BPA group, CCFs could significantly increase the serum contents of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) in males at PND 56, as well as the transcription levels of estrogen receptor alpha (ERα), steroidogenic acute regulatory protein (StAR) and Cytochrome P450 family 11, subfamily A, and member 1 (CYP11A1). CCFs also significantly inhibit the production of reactive oxygen species (ROS), reduce oxidative stress, increase mitochondrial membrane potential, and reduce sperm apoptosis. It also has a certain regulatory effect on sperm telomere length and mitochondrial DNA copy number. These results suggest that CCFs can increase reproductive hormone and receptor levels in adult males by regulating the expression of oxidative stress correlated factors, and ultimately mitigate the negative effects of BPA on sperm quality in male mice.

Trans-species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter.

Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts.

The plant vampire diaries: a historic perspective on Cuscuta research.

The angiosperm genus Cuscuta lives as an almost achlorophyllous root- and leafless holoparasite and has therefore occupied scientists for more than a century. The 'evolution' of Cuscuta research started with early studies that established the phylogenetic framework for this unusual genus. It continued to produce groundbreaking cytological, morphological, and physiological insight throughout the second half of the 20th century and culminated in the last two decades in exciting discoveries regarding the molecular basis of Cuscuta parasitism that were facilitated by the modern 'omics' tools and traceable fluorescent marker technologies of the 21st century. This review will show how present activities are inspired by those past breakthroughs. It will describe significant milestones and recurring themes of Cuscuta research and connect these to the remaining as well as newly evolving questions and future directions in this research field that is expected to sustain its strong growth in the future.

A comparative study across the parasitic plants of Cuscuta subgenus Grammica (Convolvulaceae) reveals a possible loss of the plastid genome in its section Subulatae.

MAIN CONCLUSION: Most species in Cuscuta subgenus Grammica retain many photosynthesis-related plastid genes, generally under purifying selection. A group of holoparasitic species in section Subulatae may have lost their plastid genomes entirely. The c. 153 species of plants belonging to Cuscuta subgenus Grammica are all obligate stem parasites. However, some have completely lost the ability to conduct photosynthesis while others retain photosynthetic machinery and genes. The plastid genome that primarily encodes key photosynthesis genes functions as a bellwether for how reliant plants are on primary production. This research assembles and analyses 17 plastomes across Cuscuta subgenus Grammica with the aim of characterizing the state of the plastome in each of its sections. By comparing the structure and content of plastid genomes across the subgenus, as well as by quantifying the selection acting upon each gene, we reconstructed the patterns of plastome change within the phylogenetic context for this group. We found that species in 13 of the 15 sections that comprise Grammica retain the bulk of plastid photosynthesis genes and are thus hemiparasitic. The complete loss of photosynthesis can be traced to two clades: the entire section Subulatae and a complex of three species within section Ceratophorae. We were unable to recover any significant plastome sequences from section Subulatae, suggesting that plastomes in these species are either drastically reduced or lost entirely.

Germinating seedlings and mature shoots of Cuscuta campestris respond differently to light stimuli during parasitism but not during circumnutation.

Seedlings of the parasitic plant genus Cuscuta (dodder) locate hosts by circumnutation, coil around the host near soil level and form a haustorium, establishing a primary parasitism beneath the canopy. Mature shoots elongating from the parasitic region parasitize other hosts on the upper surfaces of their canopy. Although parasitism by dodder is stimulated by blue and far-red light, and inhibited by red light, the responses to light signals during the developmental stages are not comprehensively understood. Therefore, we compared the effects of different types of light on both circumnutation and parasitism by germinating seedlings and mature shoots of Cuscuta campestris. Seedlings established parasitism under blue and far-red light, but not under red light, as has been reported repeatedly. By contrast, mature shoots exhibited coiling around the host and haustoria formation even under a red light as well as under blue and far-red light. These findings indicate that C. campestris modified its response to red light during the transition from young seedlings to mature shoots, facilitating parasitism. Light quality did not affect the circumnutation of either seedlings or mature shoots, indicating that circumnutation and the coiling movement that leads to parasitism were regulated by different environmental signals.

16S rDNA sequencing combined with metabolomics profiling with multi-index scoring method reveals the mechanism of salt-processed Semen Cuscuta in Bushen Antai mixture on kidney yang deficiency syndrome.

Kidney yang deficiency syndrome (KYDS) is a classic syndrome of traditional Chinese medicine (TCM). The salt-processed product of Semen Cuscuta (YP) is the monarch drug in Bushen Antai Mixture (BAM), can improve the reproductive dysfunction caused by KYDS, and the effect is better than that of raw products of Semen Cuscuta (SP). However, its mechanism is not completely clear yet. In this study, an integrated strategy combining untargeted metabolomics with microbiology was used to explore the mechanism of YP in the BAM improving KYDS. 16S rDNA gene sequencing showed that BAM containing YP (Y-BAM) had a significantly better regulatory effect on Desulfobacterota and Desulfovibrionaceae_unclassified than BAM containing SP (S-BAM). Untargeted metabolomics studies showed that Y-BAM significantly regulated 4 metabolites and 4 metabolic pathways. In addition, multi-index analysis showed that the effect of Y-BAM on arachidonic acid metabolism, tyrosine metabolism, purine metabolism, fructose and mannose metabolism and total metabolism was closer to that of the control group compared to S-BAM. The analysis of serum biochemical indexes showed that Y-BAM had more significant regulating effect on the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and superoxide dismutase (SOD) in serum of KYDS rats compared to S-BAM. Spearman correlation analysis showed that there was a significant correlation between intestinal microorganisms and metabolites and serum biochemical indexes. For example, Desulfovibrionaceae_unclassified was positively correlated with arachidonic acid, and negatively correlated with SOD and LH. This study suggests that YP may enhance the regulation of intestinal flora and endogenous metabolism of KYDS, so that BAM shows a better therapeutic effect on KYDS, which also reasonably explains why BAM uses Semen Cuscuta stir-baked with salt solution.