Transcriptomics and gut microbiome analysis of the edible herb Bidens pilosa as a functional feed additive to promote growth and metabolism in tilapia (Oreochromis spp.).

To reduce the use of antibiotics and chemicals in aquaculture, an edible herb, Bidens pilosa, has been selected as a multifunctional feed additive. Although there has been considerable research into the effects of B. pilosa on poultry, the wider effects of B. pilosa, particularly on the growth and gut microbiota of fish, remain largely unexplored. We aimed to investigate the interactive effects between the host on growth and the gut microbiota using transcriptomics and the gut microbiota in B. pilosa-fed tilapia. In this study, we added 0.5% and 1% B. pilosa to the diet and observed that the growth performance of tilapia significantly increased over 8 weeks of feeding. Comparative transcriptome analysis was performed on RNA sequence profiles obtained from liver and muscle tissues. Functional enrichment analysis revealed that B. pilosa regulates several pathways and genes involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, endocrine system, signal transduction, and metabolism of other amino acids. The expression of the selected growth-associated genes was validated by qRT-PCR. The qRT-PCR results indicated that B. pilosa may enhance growth performance by activating the expression of the liver igf1 and muscle igf1rb genes and inhibiting the expression of the muscle negative regulator mstnb. Both the enhancement of liver endocrine IGF1/IGF1Rb signaling and the suppression of muscle autocrine/paracrine MSTN signaling induced the expression of myogenic regulatory factors (MRFs), myod1, myog and mrf4 in muscle to promote muscle growth in tilapia. The predicted function of the gut microbiota showed several significantly different pathways that overlapped with the KEGG enrichment results of differentially expressed genes in the liver transcriptomes. This finding suggested that the gut microbiota may influence liver metabolism through the gut-liver axis in B. pilosa-fed tilapia. In conclusion, dietary B. pilosa can regulate endocrine IGF1 signaling and autocrine/paracrine MSTN signaling to activate the expression of MRFs to promote muscle growth and alter the composition of gut bacteria, which can then affect liver amino acid metabolism, carbohydrate metabolism, endocrine system, lipid metabolism, metabolism of other amino acids, and signal transduction in the host, ultimately enhancing growth performance. Our results suggest that B. pilosa has the potential to be a functional additive that can be used as an alternative to reduce antibiotic use as a growth promoter in aquaculture.

Role of arbuscular mycorrhizal fungi in lead translocation from Bidens pilosa L. plants to soil.

Bidens pilosa frequently forms a symbiotic association with arbuscular mycorrhizal fungi (AMF). This plant species can grow in Pb-polluted soils, accumulating Pb in its tissues. The aims of the study were to determine whether Pb accumulated in the tissues of B. pilosa can be transferred to the soil through AMF and to compare the role of AMF communities that have a history of exposure to the contaminant with those that have never been exposed. The experiment combined plants with and without Pb accumulated in their tissues, and inoculated with AMF collected from the rhizosphere of B. pilosa in soils contaminated and not contaminated with Pb. The results showed that AMF participate in the removal of Pb that had entered the plant and release it into the soil, as evidenced by the presence of Pb in the AMF spores and in the glomalin produced by AMF. We propose that Pb accumulation in AMF spores would be a protection mechanism that interrupts Pb uptake by the plant; however, that mechanism would not be fully exploited in detoxification, whereas the production of Pb-enriched glomalin could be an important detoxification mechanism to eliminate Pb already taken up by plants. AMF with a history of Pb exposure achieved only higher rates of root colonization, while AMF without previous exposure showed higher Pb concentration in the spores and higher glomalin production, and successfully removed Pb from both the roots and aboveground parts of the plant. The use of AMF communities not adapted to Pb may be a more effective option for microbe-mediated phytoremediation methods in which detoxification mechanisms are desirable.

Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 µM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

Analysis of the chloroplast genome and phylogenetic evolution of Bidens pilosa.

Chloroplast genomes for 3 Bidens plants endemic to China (Bidens bipinnata Linn., Bidens pilosa Linn., and Bidens alba var. radiata) have been sequenced, assembled and annotated in this study to distinguish their molecular characterization and phylogenetic relationships. The chloroplast genomes are in typical quadripartite structure with two inverted repeat regions separating a large single copy region and a small single copy region, and ranged from 151,599 to 154,478 bp in length. Similar number of SSRs and long repeats were found in Bidens, wherein mononucleotide repeats (A/T), forward and palindromic repeats were the most in abundance. Gene loss of clpP and psbD, IR expansion and contraction were detected in these Bidens plants. It seems that ndhE, ndhF, ndhG, and rpl32 from the Bidens plants were under positive selection while the majority of chloroplast genes were under purifying selection. Phylogenetic analysis revealed that 3 Bidens plants clustered together and further formed molophyletic clade with other Bidens species, indicating Bidens plants might be under radiation adaptive selection to the changing environment world-widely. Moreover, mutation hotspot analysis and in silico PCR analysis indicated that inter-genic regions of ndhD-ccsA, ndhI-ndhG, ndhF-rpl32, trnL_UAG-rpl32, ndhE-psaC, matK-rps16, rps2-atpI, cemA-petA, petN-psbM were candidate markers of molecular identification for Bidens plants. This study may provide useful information for genetic diversity analysis and molecular identification for Bidens species.

Gut microbiome helps honeybee (Apis mellifera) resist the stress of toxic nectar plant (Bidens pilosa) exposure: Evidence for survival and immunity.

Honeybee (Apis mellifera) ingestion of toxic nectar plants can threaten their health and survival. However, little is known about how to help honeybees mitigate the effects of toxic nectar plant poisoning. We exposed honeybees to different concentrations of Bidens pilosa flower extracts and found that B. pilosa exposure significantly reduced honeybee survival in a dose-dependent manner. By measuring changes in detoxification and antioxidant enzymes and the gut microbiome, we found that superoxide dismutase, glutathione-S-transferase and carboxylesterase activities were significantly activated with increasing concentrations of B. pilosa and that different concentrations of B. pilosa exposure changed the structure of the honeybee gut microbiome, causing a significant reduction in the abundance of Bartonella (p < 0.001) and an increase in Lactobacillus. Importantly, by using Germ-Free bees, we found that colonization by the gut microbes Bartonella apis and Apilactobacillus kunkeei (original classification as Lactobacillus kunkeei) significantly increased the resistance of honeybees to B. pilosa and significantly upregulated bee-associated immune genes. These results suggest that honeybee detoxification systems possess a level of resistance to the toxic nectar plant B. pilosa and that the gut microbes B. apis and A. kunkeei may augment resistance to B. pilosa stress by improving host immunity.

Effects of a mucoadhesive phytomedicine (Curcuma longa L. and Bidens pilosa L.) on radiotherapy-induced oral mucositis and quality of life of patients undergoing head and neck cancer treatment: randomized clinical trial.

PURPOSE: To assess the effect of a mucoadhesive herbal medicine containing curcuminoids and a glycerinated extract of Bidens pilosa L. (FITOPROT) in association with photobiomodulation (PBM) therapy and a Preventive Oral Care Program (POCP) compared to PBM and POCP in the treatment of radiotherapy (RT)-induced oral mucositis (ROM) and in the quality of life of these patients. METHODS: A double-blind clinical trial was performed with head and neck cancer patients undergoing RT or chemoradiotherapy. Participants were randomized into two groups: Group 1 (n=27): PBM and POCP; and Group 2 (n=25): PBM, POCP and FITOPROT. The PBM protocol was daily irradiation, 660 nm, 25mW, 0.25 J/point from the first until the last day of RT. The FITOPROT was used as mouthwash twice a day. ROM was evaluated based on the scales of the World Health Organization and National Cancer Institute. The quality of life was evaluated using the University of Washington Questionnaire, OHIP-14 and Patient-Reported Oral Mucositis Symptom Scale. The MMAS-8 questionnaire was used to evaluated the adherence to POCP and FITOPROT. Data were collected at baseline, 7th, 14th, 21st, and 30th RT sessions. RESULTS: No statistical differences were found between the groups for the ROM evaluation. Both groups experienced worsening of the quality of life during the RT. No statistically significant differences between groups were observed for any of the instruments evaluated. CONCLUSION: The results suggest that PBM associated with FITOPROT and POCP control the severity of ROM and stabilize the QoL of patients with head and neck cancer. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials (ReBEC-RBR-9vddmr; UTN code: U1111-1193-2066), registered in August 8th, 2017.

(E)-7-phenyl-2-heptene-4,6-diyn-1-ol from Bidens pilosa as a repellent against isopods.

The development of repellents as alternatives to insecticides has expanded in recent years. However, their use in isopod pest control is limited. To develop an isopod repellent, a plant extract library from wild plants native to the Kochi Prefecture was screened for repellent activity against pillbugs, and 82 samples (87%) exhibited repellent activity. Among them, (E)-7-phenyl-2-heptene-4,6-diyn-1-ol was isolated and identified as a repellent from the root of Bidens pilosa. It had a half-maximal effective concentration of 0.20 µm, with a strong repellency. A study of the structure-activity relationship to (E)-7-phenyl-2-heptene-4,6-diyn-1-ol revealed that the presence of a hydroxyl group and an aromatic at both ends of the length of the seven-carbon chain is important for the expression of repellency. These results can potentially lead to a new repellent of phenylalkyl alcohol.

Overall metabolic network analysis of urine in hyperlipidemic rats treated with Bidens bipinnata L.

Hyperlipidemia has been highlighted as one of the most prominent and global chronic conditions nowadays. Bidens bipinnata L. (BBL), a folk medicine in contemporary China, has efficacy in the treatment of hyperlipidemia (HLP) in China. Although some physiological and pathological function parameters of hyperlipidemia have been investigated, little information about the changes in small metabolites in biofluids has been reported. In the present study, global metabolic profiling with high-performance liquid chromatography-linear ion trap/Orbitrap high-resolution mass spectrometry (HPLC-LTQ/Orbitrap MS) combined with a pattern recognition method was performed to discover the underlying lipid-regulating mechanisms of BBL on hyperlipidemic rats induced by high-fat diet (HFD). The total of four metabolites, up- or down-regulated (p < 0.05 or 0.01), were identified and contributed to the progression of hyperlipidemia. These promising identified biomarkers underpin the metabolic pathway, including glyoxylate and dicarboxylate metabolism, the TCA cycle, sphingolipid metabolism and purine metabolism. They are disturbed in hyperlipidemic rats, and are identified using pathway analysis with MetPA. The altered metabolite indices could be regulated closer to normal levels after BBL intervention. The results demonstrated that urinary metabolomics is a powerful tool in the clinical diagnosis and treatment of hyperlipidemia to provide information on changes in metabolite pathways.

Comparative study on plant growth-promoting bacterial inoculation by irrigation and spraying for promoting Bidens pilosa L. phytoremediation of cadmium-contaminated soil.

A field study was conducted to compare FM-1 inoculation by irrigation and spraying for promoting Bidens pilosa L. phytoremediation of cadmium (Cd)-contaminated soil. Cascading relationships between bacterial inoculation by irrigation and spraying and soil properties, plant growth-promoting traits, plant biomass and Cd concentrations in Bidens pilosa L. were explored based on the partial least squares path model (PLS-PM). The results indicated that inoculation with FM-1 not only improved the rhizosphere soil environment of B. pilosa L. but also increased the Cd extracted from the soil. Moreover, Fe and P in leaves play vital roles in promoting plant growth when FM-1 is inoculated by irrigation, while Fe in leaves and stems plays a vital role in promoting plant growth when FM-1 is inoculated by spraying. In addition, FM-1 inoculation decreased the soil pH by affecting soil dehydrogenase and oxalic acid in cases with irrigation and Fe in roots in cases with spraying. Thus, the soil bioavailable Cd content increased and promoted Cd uptake by Bidens pilosa L. To address Cd-induced oxidative stress, Fe in leaves helped to convert GSH into PCs, which played a vital role in ROS scavenging when FM-1 was inoculated by irrigation. The soil urease content effectively increased the POD and APX activities in the leaves of Bidens pilosa L., which helped alleviate Cd-induced oxidative stress when FM-1 was inoculated by spraying. This study compares and illustrates the potential mechanism by which FM-1 inoculation can improve the phytoremediation of Cd-contaminated soil by Bidens pilosa L., suggesting that FM-1 inoculation by irrigation and spraying is useful in the phytoremediation of Cd-contaminated sites.

The co-phytotoxicity of two Asteraceae invasive plants Solidago canadensis L. and Bidens pilosa L. with different invasion degrees.

The phytotoxicity of invasive plants (IPS) has been identified as one of the main factors influencing their invasion success. The invasion of IPS can occur to varying degrees in the habitats. Two IPS can invade one habitat. This study aimed to evaluate the mono- and co-phytotoxicity of two Asteraceae IPS Solidago canadensis L. and Bidens pilosa L. with different invasion degrees (including light invasion (relative abundance <50%) and heavy invasion (relative abundance ≥50%)) on the horticultural Asteraceae species Lactuca sativa L., through a hydroponic experiment conducted on 9 cm Petri dishes. Leaf extracts of the two IPS can cause significant mono- and co-phytotoxicity. The mono- and co-phytotoxicity of the two IPS were concentration-dependent. The mono-phytotoxicity of S. canadensis was significantly increased with increasing invasion degree, but the opposite was true for the mono-phytotoxicity of B. pilosa. Leaf extracts of B. pilosa with light invasion caused stronger phytotoxicity than those of S. canadensis with light invasion. There may be an antagonistic effect for the co-phytotoxicity caused by mixed leaf extracts of the two IPS compared with those of either S. canadensis or B. pilosa. The phytotoxicity of the two IPS on the growth performance of neighboring plants may play a more important role in their mono-invasion than in their co-invasion. The phytotoxicity appeared to affect the growth performance of S. canadensis individuals more significantly when the invasion was heavy, while the growth performance of B. pilosa individuals seemed to be more influenced by phytotoxicity when the invasion was light. Consequently, the concentration of leaf extracts of IPS, the invasion degree of IPS, the species identity of IPS, and the species number of IPS modulated the mono- and co-phytotoxicity of the two IPS.

Anti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse Model of Amyotrophic Lateral Sclerosis and Lipopolysaccharide-Stimulated BV-2 Microglia.

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-ß and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Polyacetylene Isomers Isolated from Bidens pilosa L. Suppress the Metastasis of Gastric Cancer Cells by Inhibiting Wnt/ß-Catenin and Hippo/YAP Signaling Pathways.

(E)-7-Phenyl-2-hepten-4,6-diyn-1-ol (1) and (Z)-7-Phenyl-2-hepten-4,6-diyn-1-ol (2) are isomeric natural polyacetylenes isolated from the Chinese medicinal plant Bidens pilosa L. This study first revealed the excellent anti-metastasis potential of these two polyacetylenes on human gastric cancer HGC-27 cells and the distinctive molecular mechanisms underlying their activities. Polyacetylenes 1 and 2 significantly inhibited the migration, invasion, and adhesion of HGC-27 cells at their non-toxic concentrations in a dose-dependent manner. The results of a further mechanism investigation showed that polyacetylene 1 inhibited the expressions of Vimentin, Snail, ß-catenin, GSK3ß, MST1, YAP, YAP/TAZ, and their phosphorylation, and upregulated the expression of E-cadherin and p-LATS1. In addition, the expressions of various downstream metastasis-related proteins, such as MMP2/7/9/14, c-Myc, ICAM-1, VCAM-1, MAPK, p-MAPK, Sox2, Cox2, and Cyr61, were also suppressed in a dose-dependent manner. These findings suggested that polyacetylene 1 exhibited its anti-metastasis activities on HGC-27 cells through the reversal of the EMT process and the suppression of the Wnt/ß-catenin and Hippo/YAP signaling pathways.

A Bidens pilosa L. Non-Polar Extract Modulates the Polarization of Human Macrophages and Dendritic Cells into an Anti-Inflammatory Phenotype.

Different communities around the world traditionally use Bidens pilosa L. for medicinal purposes, mainly for its anti-inflammatory, antinociceptive, and antioxidant properties; it is used as an ingredient in teas or herbal medicines for the treatment of pain, inflammation, and immunological disorders. Several studies have been conducted that prove the immunomodulatory properties of this plant; however, it is not known whether the immunomodulatory properties of B. pilosa are mediated by its ability to modulate antigen-presenting cells (APCs) such as macrophages (MØs) and dendritic cells (DCs) (through polarization or the maturation state, respectively). Different polar and non-polar extracts and fractions were prepared from the aerial part of B. pilosa. Their cytotoxic and immunomodulatory effects were first tested on human peripheral blood mononuclear cells (PBMCs) and phytohemagglutinin (PHA)-stimulated PBMCs, respectively, via an MTT assay. Then, the non-cytotoxic plant extracts and fractions that showed the highest immunomodulatory activity were selected to evaluate their effects on human MØ polarization and DC maturation (cell surface phenotype and cytokine secretion) through multiparametric flow cytometry. Finally, the chemical compounds of the B. pilosa extract that showed the most significant immunomodulatory effects on human APCs were identified using gas chromatography coupled with mass spectrometry. The petroleum ether extract and the ethyl acetate and hydroalcoholic fractions obtained from B. pilosa showed low cytotoxicity and modulated the PHA-stimulated proliferation of PBMCs. Furthermore, the B. pilosa petroleum ether extract induced M2 polarization or a hybrid M1/M2 phenotype in MØs and a semi-mature status in DCs, regardless of exposure to a maturation stimulus. The immunomodulatory activity of the non-polar (petroleum ether) extract of B. pilosa on human PBMC proliferation, M2 polarization of MØs, and semi-mature status in DCs might be attributed to the low-medium polarity components in the extract, such as phytosterol terpenes and fatty acid esters.

Effect of temperature and water potential on the germination of seeds from three different populations of Bidens pilosa as a potential Cd hyperaccumulator.

BACKGROUND: Bidens pilosa L., an annual herb, has recently been shown to be a potential Cd-hyperaccumulating plant. The germination characteristics of B. pilosa have been documented, while the difference among populations remains unclear. Understanding variability in seed germination among populations is crucial for determining which populations to use for soil remediation programs. RESULTS: Present study was conducted to compare the requirements of temperature and water potential for germination of B. pilosa cypselae (the central type, hereafter seeds) from three populations using the thermal time, hydrotime, and hydrothermal time models. Seeds of three populations were incubated at seven constant temperatures (8, 12, 15, 20, 25, 30, and 35 °C) and at each of four water potentials (0, -0.3, -0.6, and -0.9 MPa). The results showed that germination percentage and rate of B. pilosa seeds were significantly by population, temperature, water potential and their interaction except for the interaction of population and water potential. Seeds from Danzhou population displayed a higher base temperature (Tb) for germination than those from Guilin and Baoshan population, however the ceiling temperature (Tc) had no consistent level among the populations but varied according to the water potential. In addition, the median base water potential [ψb(50)] for germination of seeds from Danzhou population was higher than that for seeds from Baoshan and Guilin population at low temperatures (< 25 °C), which was opposite at high temperatures (≥ 25 °C). CONCLUSION: Seed germination requirements of B. pilosa on temperature and water differed significantly among populations. Differences in seed germination among populations may be complicated, which could not be simply explained by the temperature and rainfall conditions where the seeds were produced as previously reported. The results suggested that programme management should consider variation in seed germination traits when select which population could be applied to what kind of target remediation sites.

Identification and genome analysis of a tomato zonate spot virus isolate from Bidens pilosa.

Bidens pilosa is a weed species that invades crop areas in tropical and subtropical regions. To date, only two potyviruses have been reported to infect B. pilosa. Here, we report the complete genome sequence of a tomato zonate spot tospovirus (TZSV) isolate from Bidens named TZSV-Bidens. The tripartite RNA of the TZSV-Bidens genome contains L, M, and S segments that are 8912, 4724, and 2997 nt in length, respectively. The genome contains five open reading frames (ORFs), with 92.23-95.01% amino acid sequence identity to the TZSV-YN isolate. Phylogenetic analysis based on amino acid sequences of members of the family Tospoviridae showed that TZSV-Bidens was grouped into a well-supported Eurasian cluster. The intergenic regions (IGRs) of the M and S RNAs are among the most variable regions and are far shorter than those of the TZSV-YN reference genome.

A Genome for Bidens hawaiensis: A Member of a Hexaploid Hawaiian Plant Adaptive Radiation.

The plant genus Bidens (Asteraceae or Compositae; Coreopsidae) is a species-rich and circumglobally distributed taxon. The 19 hexaploid species endemic to the Hawaiian Islands are considered an iconic example of adaptive radiation, of which many are imperiled and of high conservation concern. Until now, no genomic resources were available for this genus, which may serve as a model system for understanding the evolutionary genomics of explosive plant diversification. Here, we present a high-quality reference genome for the Hawai'i Island endemic species B. hawaiensis A. Gray reconstructed from long-read, high-fidelity sequences generated on a Pacific Biosciences Sequel II System. The haplotype-aware, draft genome assembly consisted of ~6.67 Giga bases (Gb), close to the holoploid genome size estimate of 7.56 Gb (±0.44 SD) determined by flow cytometry. After removal of alternate haplotigs and contaminant filtering, the consensus haploid reference genome was comprised of 15 904 contigs containing ~3.48 Gb, with a contig N50 value of 422 594. The high interspersed repeat content of the genome, approximately 74%, along with hexaploid status, contributed to assembly fragmentation. Both the haplotype-aware and consensus haploid assemblies recovered >96% of Benchmarking Universal Single-Copy Orthologs. Yet, the removal of alternate haplotigs did not substantially reduce the proportion of duplicated benchmarking genes (~79% vs. ~68%). This reference genome will support future work on the speciation process during adaptive radiation, including resolving evolutionary relationships, determining the genomic basis of trait evolution, and supporting ongoing conservation efforts.

Functional and Mechanistic Studies of Two Anti-coccidial Herbs, Bidens pilosa and Artemisia indica.

Currently, antibiotics are commonly used to treat coccidiosis, a severe protozoal disease in chickens. However, due to growing concerns about the antibiotic residue in meat and eggs, phytogenic formulations are becoming an attractive approach to manage this disease. In this study, we investigated the anti-coccidial function and mechanism of phytogenic formulations composed of Bidens pilosa, Artemisia indica, and both used in combination. We found that these formulations increased the survival rate and reduced body weight loss, the feed conversion ratio, oocyst excretion, bloody stools, and gut lesions of chickens. Mechanistic studies showed that A. indica, but not B. pilosa, reduced the survival of Eimeria oocysts. Accordingly, they both inhibited oocyst sporulation and sporozoite invasion into Madin-Darby bovine kidney (MDBK) cells. Overall, we demonstrate that these formulations protect chickens against coccidiosis. Moreover, a combination of B. pilosa and A. indica has an additive effect on coccidiosis control and growth performance in chickens compared to either one used alone.

Bruceine D may affect the phenylpropanoid biosynthesis by acting on ADTs thus inhibiting Bidens pilosa L. seed germination.

Bruceine D is a natural quassinoid, which was successfully isolated in our research group from the residue of Brucea javanica (L.) seeds. Our previous research showed that Bruceine D prevented Bidens pilosa L. seed germination by suppressing the activity of key enzymes and the expression levels of key genes involved in the phenylpropanoid biosynthesis pathway. In this study, integrated analyses of non-targeted metabolomic and transcriptomic were performed. A total of 356 different accumulated metabolites (DAMs) were identified, and KEGG pathway analyses revealed that most of these DAMs were involved in phenylpropanoid biosynthesis. The decreased expression of ADTs and content of L-phenylalanine implicates that Bruceine D may suppress the downstream phenylpropanoid biosynthesis pathway by disrupting primary metabolism, that is, the phenylalanine biosynthesis pathway, thus inhibiting the final products, resulting in the interruption of B. pilosa seed germination. These results suggest that Bruceine D may inhibit the B. pilosa seed germination by suppressing phenylpropanoid biosynthesis through acting on ADTs.

A new dihydroflavone and a new polyacetylene glucoside from Bidens parviflora.

A new dihydroflavone, 2(S)-isookanin-4'-methoxy-8-O-ß-D-glucopyranoside (1), and a new polyacetylene glucoside, (10S)-tridecane-2E-ene-4,6,8-triyne-1-ol-10-O-ß-D-glucopyranoside (2), along with seven known compounds (3-9), were isolated from the herb of Bidens parviflora Willd. The structures of all the extracted compounds were elucidated by HR-ESI-MS, 1 D and 2 D NMR spectra, as well as circular dichroism (CD).

Elucidation of enzymes involved in the biosynthetic pathway of bioactive polyacetylenes in Bidens pilosa using integrated omics approaches.

Polyacetylene compounds from Bidens pilosa are known to have several pharmacological activities. In this study, we identified major genes encoding enzymes involved in the biosynthesis of polyacetylene in B. pilosa. Seven polyacetylene metabolites present in B. pilosa leaves were induced by methyl jasmonate (MeJA) treatment and physical wounding. Transcriptome analysis via high-throughput sequencing revealed 39 202 annotated gene fragment sequences. A DNA microarray established by the 39 202 annotated genes was used to profile gene expression in B. pilosa leaf and root tissues. As no polyacetylene compounds were found in roots, the gene expression pattern in root tissue was used as a negative control. By subtracting MeJA-induced genes in roots, we obtained 1216 genes in leaves showing an approximate three-fold increase in expression post-MeJA treatment. Nine genes encoding enzymes with desaturation function were selected for confirmation of expression by qRT-PCR. Among them, two genes, BPTC030748 and BPTC012564, were predicted to encode Δ12-oleate desaturase (OD) and Δ12-fatty acid acetylenase (FAA), respectively. In B. pilosa leaves, RNAi knock-down concomitantly decreased, while virus-mediated transient overexpression of either gene elevated polyacetylene content. In summary, we demonstrate that two important enzymes, Δ12-oleate desaturase and Δ12-fatty acid acetylenase, involved in desaturation of linear fatty acid precursors play a role in polyacetylene biosynthesis in an important medicinal plant, Bidens pilosa.