Genome assemblies of Vigna reflexo-pilosa (créole bean) and its progenitors, Vigna hirtella and Vigna trinervia, revealed homoeolog expression bias and expression-level dominance in the allotetraploid.

Vigna reflexo-pilosa (créole bean) is a wild legume belonging to the subgenus Ceratoropis and is widely distributed in Asia. Créole bean is the only tetraploid species in the genus Vigna, and it has been shown to derive from the hybridization of Vigna hirtella and Vigna trinervia. In this study, we combined the long-read PacBio technology with the chromatin contact mapping (Hi-C) technique to obtain a chromosome-level assembly of V. reflexo-pilosa. The final assembly contained 998,724,903 bases with an N50 length of 42,545,650 bases. Our gene prediction recovered 99.4% of the highly conserved orthologs based on the BUSCO analysis. To investigate homoeolog expression bias and expression level dominance in the tetraploid, we also sequenced and assembled the genomes of its progenitors. Overall, the majority of the homoeolog pairs (72.9%) displayed no expression bias, and among those that exhibited biased expression, 16.3% showed unbalanced homoeolog expression bias toward the V. trinervia subgenome. Moreover, 41.2% and 36.2% of the expressed gene pairs exhibited transgressive expression and expression level dominance, respectively. Interestingly, the genome-wide expression level dominance in the tetraploid was biased toward the V. trinervia subgenome. The analysis of methylation patterns also revealed that the average methylation levels in coding regions were higher in the V. hirtella subgenome than those in the V. trinervia subgenome. The genomic/transcriptomic resources for these three species are useful not only for the development of elite cultivars in Vigna breeding programs but also to researchers studying comparative genomics and investigating genomic/epigenomic changes following polyploid events.

A new prenylated coumestan from the roots of Campylotropis hirtella.

A new prenylated coumestan, campylohirtin A (1), along with fifteen phenolic known compounds (2‒16) and four other known compounds (17‒20), was obtained from the 95% ethanol extract of roots of Campylotropis hirtella. Their structures were elucidated based on extensive spectroscopic analysis (1 D and 2 D-NMR, MS, UV and IR). In vitro antimalarial activities of compounds 1-3, 5-14 and 16 were evaluated by ß-hematin formation inhibition assay. Compared with the positive control chloroquine diphosphate, compounds 8, 11 and 16 exhibited strong antimalarial activity with the IC50 values of 69.9, 33.2 and 75.4 µM, respectively. Compounds 1-3, 5-7 and 12 showed moderate antimalarial activities with IC50 values ranging from 134.6 µM to 578.6 µM.[Formula: see text].

Do Host Plant and Associated Ant Species Affect Microbial Communities in Myrmecophytes?

Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus to build galleries on their host-plant stems. Allomerus-inhabited domatia, thus, might be a rich arena for microbes associated with the ants, the plant, and the fungus. We investigated the microbial communities present in domatia colonised by four arboreal ants: Allomerus decemarticulatus, A. octoarticulatus, A. octoarticulatus var. demerarae, and the non-fungus growing plant-ant Azteca sp. cf. depilis, inhabiting Hirtella physophora or Cordia nodosa in French Guiana. We hypothesized that the microbial community will differ among these species. We isolated microorganisms from five colonies of each species, sequenced the 16S rRNA or Internal TranscribedSpacer (ITS) regions, and described both the alpha and beta diversities. We identified 69 microbial taxa, which belong to five bacterial and two fungal phyla. The most diverse phyla were Proteobacteria and Actinobacteria. The microbial community of Azteca cf. depilis and Allomerus spp. differed in composition and richness. Geographical distance affected microbial communities and richness but plant species did not. Actinobacteria were only associated with Allomerus spp.

Convergent structure and function of mycelial galleries in two unrelated Neotropical plant-ants.

The construction process and use of galleries by Azteca brevis (Myrmicinae: Dolichoderinae) inhabiting Tetrathylacium macrophyllum (Salicaceae) were compared with Allomerus decemarticulatus (Myrmicinae: Solenopsidini) galleries on Hirtella physophora (Chrysobalanaceae). Though the two ant species are phylogenetically distant, the gallery structure seems to be surprisingly similar and structurally convergent: both are pierced with numerous holes and both ant species use Chaetothyrialean fungi to strengthen the gallery walls. Al. decemarticulatus is known to use the galleries for prey capture and whether this is also the case for Az. brevis was tested in field experiments. We placed Atta workers as potential prey/threat on the galleries and recorded the behaviour of both ant species. We found considerable behavioural differences between them: Al. decemarticulatus was quicker and more efficient at capture than was Az. brevis. While most Atta workers were captured after the first 5 min by Al. decemarticulatus, significantly fewer were captured by Az. brevis even after 20 min. Moreover, the captured Atta were sometimes simply discarded and not taken to the nest by Az. brevis. As a consequence, the major function of the galleries built by Az. brevis may, therefore, be defense against intruders in contrast to Al. decemarticulatus which uses them mainly for prey capture. This may be due to a higher need for protein in Al. decemarticulatus compared to coccid-raising Az. brevis.

Exploring fungus-plant N transfer in a tripartite ant-plant-fungus mutualism.

Background and Aims: The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods: Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a 15N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added 15N from the fungus to plant tissues. Key Results: The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The 15N provisioning of the fungus in the galleries resulted in significant enrichment of the 15N signature of the plant's leaves 1 d after the 15N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions: This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from 15N-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.

Trade-offs in an ant-plant-fungus mutualism.

Species engaged in multiple, simultaneous mutualisms are subject to trade-offs in their mutualistic investment if the traits involved in each interaction are overlapping, which can lead to conflicts and affect the longevity of these associations. We investigate this issue via a tripartite mutualism involving an ant plant, two competing ant species and a fungus the ants cultivate to build galleries under the stems of their host plant to capture insect prey. The use of the galleries represents an innovative prey capture strategy compared with the more typical strategy of foraging on leaves. However, because of a limited worker force in their colonies, the prey capture behaviour of the ants results in a trade-off between plant protection (i.e. the ants patrol the foliage and attack intruders including herbivores) and ambushing prey in the galleries, which has a cascading effect on the fitness of all of the partners. The quantification of partners' traits and effects showed that the two ant species differed in their mutualistic investment. Less investment in the galleries (i.e. in fungal cultivation) translated into more benefits for the plant in terms of less herbivory and higher growth rates and vice versa. However, the greater vegetative growth of the plants did not produce a positive fitness effect for the better mutualistic ant species in terms of colony size and production of sexuals nor was the mutualist compensated by the wider dispersal of its queens. As a consequence, although the better ant mutualist is the one that provides more benefits to its host plant, its lower host-plant exploitation does not give this ant species a competitive advantage. The local coexistence of the ant species is thus fleeting and should eventually lead to the exclusion of the less competitive species.

Human neutrophil elastase inhibitory potential of flavonoids from Campylotropis hirtella and their kinetics.

Campylotropis hirtella is used as a food supplement in the subtropical region of China. In an intensive hunt for human neutrophil elastase inhibitors, we isolated eight flavonoids from C. hirtella three of which (1-3) emerged to be elastase inhibitors. Geranylated flavonoids (1-3) displayed significant inhibitory activity with IC50s between 8.5 and 30.8 µM. The most striking example was geranylated isofavanone 3 that inhibited elastase significantly (IC50 = 30.8 µM) but its parent compound (dalbergioidin) and isoflavone analog (5) were inactive (IC50 > 200 µM). Compounds (1-3) displayed different kinetic mechanisms (noncompetitive, competitive, and mixed type, respectively) that were dependent upon the parent skeleton. The competitive inhibitor, isoflavan-3-ol-4-one 2 manifested an inhibition of isomerization profile for elastase with kinetic parameters K5 = 0.0386 M-1S-1, K6 = 0.0244 µM-1S-1 and Kiapp = 16.3427 µM. The specific identification of metabolites was accomplished by LC-DAD-ESI/MS that was also used to analyze abundance of active components (1-3) within the plant.

A new potent immunosuppressive isoflavanonol from Campylotropis hirtella.

Four new flavonoids were isolated from Campylotropis hirtella and these are a chromone and a 2H-chromene, an isoflavone and an isoflavanonol. The structures of these compounds were elucidated by extensive spectroscopic measurements. All of the compounds were assessed for immunosuppressive activity. Compound 4 showed very strong T lymphocyte suppression activity (IC50: 0.13 µM) and potent B lymphocyte suppression activity (IC50: 0.26 µM). Due to its potent immunosuppressive activity and lower cytotoxicity, further structure-activity studies will be pursued on this compound.

Highly potent tyrosinase inhibitor, neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking.

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson's disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 µg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC50 of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC50=500 nM), significantly. Another potent inhibitor 1 (IC50=2.9 µM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: Ki(app)=1.48 nM, k3=0.0033 nM(-1) min(-1) and k4=0.0049 min(-1). Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 µM of IC50. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.

New flavonoids from Campylotropis hirtella with immunosuppressive activity.

In an effort to identify natural compounds with immunosuppressive activity, nine new flavonoids, including one isoflav-3-ene derivative (1), one coumaronochromone (2), two isoflavanones (3, 4), one isoflavone derivative (6), one isoflavone (7), three flavonols (8, 9, 10), as well as one known compound, hydroisoflavone C (5), were isolated from the roots of Campylotropis hirtella. The structures of these compounds were elucidated by extensive spectroscopic measurements. All of the compounds were assessed for immunosuppressive activity. Among the isolates, compound 2 showed good inhibitory activity against mitogen-induced splenocyte proliferation with an IC50 of 0.28 µM and relatively low cytotoxicity.

Chrysobalanaceae: traditional uses, phytochemistry and pharmacology

Chrysobalanaceae is a family composed of seventeen genera and about 525 species. In Africa and South America some species have popular indications for various diseases such as malaria, epilepsy, diarrhea, inflammations and diabetes. Despite presenting several indications of popular use, there are few studies confirming the activities of these species. In the course of evaluating the potential for future studies, the present work is a literature survey on databases of the botanical, chemical, biological and ethnopharmacological data on Chrysobalanaceae species published since the first studies that occurred in the 60's until the present day.

Cheating the cheater: domatia loss minimizes the effects of ant castration in an Amazonian ant-plant.

We studied the relationship between Hirtella myrmecophila (Chrysobalanaceae), a common but little-studied Amazonian ant-plant that produces leaf-pouches as domatia, and its obligate ant partner, Allomerus octoarticulatus. Field observations revealed that H. myrmecophila drops domatia from older leaves, a characteristic that is unique among myrmecophytes. The physiological mechanism for abortion of domatia is currently unknown, but this characteristic allows for the existence, within the same plant, of branches with and without ants. Older branches generally bear only old leaves with no domatia and therefore have no ants, whereas younger branches have leaves of various ages. Ants forage mainly on new leaves, and experimental removal of ants showed that A. octoarticulatus is crucial for defense of these leaves against insect herbivores. However, A. octoarticulatus also acts as a castration parasite, severing the plant's inflorescences. Mature flowers and fruits were only found on older branches with no ants, and flower production was 8 times greater on plants whose ants were experimentally removed than on control plants. Given the reproductive costs inflicted by its mutualistic partner, we suggest that abortion of domatia is a strategy developed by H. myrmecophila to minimize the effects of cheating by A. octoarticulatus. These results support the view that evolutionary conflicts of interest between mutualistic species often impose selection for cheating on the partner, as well as for mechanisms to retaliate or to prevent super-exploitation. Opposing selection pressures, operating independently on the two partners, probably help to maintain the evolutionary stability of this mutualistic relationship.