Functional evidence supports adaptive plant chemical defense along a geographical cline.

Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host's range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed (Asclepias syriaca) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na+/K+-ATPase); there was little variation among compounds in inhibition of an unadapted Na+/K+-ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltus. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.

Testing the selective sequestration hypothesis: Monarch butterflies preferentially sequester plant defences that are less toxic to themselves while maintaining potency to others.

Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na+ /K+ -ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na+ /K+ -ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenic Drosophila that were CRISPR-edited with the monarch's Na+ /K+ -ATPase. Thus, the monarch's selective sequestration appears to reduce self-harm while maintaining protection from enemies.

Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest.

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus-growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species-specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.

Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra.

Driven by a necessity for confident molecular identification at high spatial resolution, a new time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem mass spectrometry (tandem MS) imaging instrument has been recently developed. In this paper, the superior MS/MS spectrometry and imaging capability of this new tool is shown for natural product study. For the first time, via in situ analysis of the bioactive metabolites rubrynolide and rubrenolide in Amazonian tree species Sextonia rubra (Lauraceae), we were able both to analyze and to image by tandem MS the molecular products of natural biosynthesis. Despite the low abundance of the metabolites in the wood sample(s), efficient MS/MS analysis of these γ-lactone compounds was achieved, providing high confidence in the identification and localization. In addition, tandem MS imaging minimized the mass interferences and revealed specific localization of these metabolites primarily in the ray parenchyma cells but also in certain oil cells and, further, revealed the presence of previously unidentified γ-lactone, paving the way for future studies in biosynthesis.

Correction: Fluorescent natural products as probes and tracers in biology.

Correction for 'Fluorescent natural products as probes and tracers in biology' by Romain Duval et al., Nat. Prod. Rep., 2017, DOI: 10.1039/c6np00111d.

Fluorescent natural products as probes and tracers in biology.

Covering: 1985 up to the end of 2016Fluorescence is a remarkable property of many natural products in addition to their medicinal and biological values. Herein, we provide a review on these peculiar secondary metabolites to stimulate prospecting of them as original fluorescent tracers, endowed with unique photophysical properties and with applications in most fields of biology. The compounds are spectrally categorized (i.e. fluorescing from violet to the near infra-red) and further structurally classified within each category. Natural products selected for their high impact in modern fluorescence-based biological studies are highlighted throughout the article. Finally, we discuss aspects of chemical ecology where fluorescent natural products might have key evolutionary roles and thus open new research directions in the field.

Tandem Biocatalysis Unlocks the Challenging de Novo Production of Plant Natural Products.

Intimate partnership: Knowledge of the biocatalytic cascades in different cellular compartments is limited, but deciphering these systems in nature can be used to inspire synthetic strategies. Two studies report new insights into the biosynthesis of alkaloids and sesterterpenoids in plants. This highlight presents these novel biotransformations to illustrate how tandem biocatalysis can impact the future of natural product production.

Revisiting Previously Investigated Plants: A Molecular Networking-Based Study of Geissospermum laeve.

Three new monoterpene indole alkaloids (1-3) have been isolated from the bark of Geissospermum laeve, together with the known alkaloids (-)-leuconolam (4), geissolosimine (5), and geissospermine (6). The structures of 1-3 were elucidated by analysis of their HRMS and NMR spectroscopic data. The absolute configuration of geissolaevine (1) was deduced from the comparison of experimental and theoretically calculated ECD spectra. The isolation workflow was guided by a molecular networking-based dereplication strategy using an in-house database of monoterpene indole alkaloids. In addition, five known compounds previously undescribed in the Geissospermum genus were dereplicated from the G. laeve alkaloid extract network and were assigned with various levels of identification confidence. The antiparasitic activities against Plasmodium falciparum and Leishmania donovani as well as the cytotoxic activity against the MRC-5 cell line were determined for compounds 1-5.

Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay.

BACKGROUND: A major requirement for malaria elimination is the development of transmission-blocking interventions. In vitro transmission-blocking bioassays currently mostly rely on the use of very few Plasmodium falciparum reference laboratory strains isolated decades ago. To fill a piece of the gap between laboratory experimental models and natural systems, the purpose of this work was to determine if culture-adapted field isolates of P. falciparum are suitable for in vitro transmission-blocking bioassays targeting functional maturity of male gametocytes: exflagellation. METHODS: Plasmodium falciparum isolates were adapted to in vitro culture before being used for in vitro gametocyte production. Maturation was assessed by microscopic observation of gametocyte morphology over time of culture and the functional viability of male gametocytes was assessed by microscopic counting of exflagellating gametocytes. Suitability for in vitro exflagellation-blocking bioassays was determined using dihydroartemisinin and methylene blue. RESULTS: In vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia. Functional maturity of male gametocytes was assessed by exflagellation observations and all four isolates could be used in exflagellation-blocking bioassays with adequate response to methylene blue and dihydroartemisinin. CONCLUSION: This work shows that in vitro culture-adapted P. falciparum field isolates of different genetic background, from South America and Southeast Asia, can successfully be used for bioassays targeting the male gametocyte to gamete transition, exflagellation.

Isolation of Guttiferones from Renewable Parts of Symphonia globulifera by Centrifugal Partition Chromatography.

The aim of this study was to investigate the species Symphonia globulifera, a source of polycyclic polyprenylated acyl phloroglucinols such as guttiferone A, which is known to exhibit a variety of biological activities including noticeable antileishmanial properties. Our goal was the identification and the quantification of guttiferone A in different renewable parts of S. globulifera and its preparative isolation. To the best of our knowledge, there is no data concerning its mechanism of action. Consequently, it is particularly interesting to isolate it in gram quantities in order to establish structure activity relationship studies. After performing high-performance liquid chromatography profiles detecting the presence of guttiferone A and proceeding to its quantification, a centrifugal partition chromatography methodology using a two-phase solvent system of cyclohexane/ethyl acetate/methanol/water (20 :  1 :  20 : 1, v/v/v/v) was applied to each extract. In conclusion, a centrifugal partition chromatography system has been developed to ensure a fast, reliable, and scalable way to isolate, with a high level of purity, guttiferone A from five renewable parts of S. globulifera. Moreover, this methodology can be extended to the isolation of other polycyclic polyprenylated acyl phloroglucinols such as guttiferones B, C, and D.

Route of exposure to veterinary products in bees: Unraveling pasture's impact on avermectin exposure and tolerance in stingless bees.

Deforestation rapidly increases in tropical regions, primarily driven by converting natural habitats into pastures for extensive cattle ranching. This landscape transformation, coupled with pesticide use, are key drivers of bee population decline. Here, we investigate the impact of pasture-dominated landscapes on colony performance, pesticide exposure, and insecticide sensitivity of the stingless bee Tetragonisca angustula. We monitored 16 colonies located in landscapes with varying proportions of pasture. We collected bee bread for pesticide and palynological analysis. We found a positive correlation between pollen diversity and colony growth, with no effect of the proportion of pasture in the landscape. In contrast, we detected prevalent and hazardous concentrations of the insecticide abamectin (9.6-1,856 µg/kg) in bee bread, which significantly increased with a higher proportion of pasture. Despite the abamectin exposure, the bee colonies displayed no adverse effects on their growth, indicating a potential tolerance response. Further investigations revealed that bees from sites with higher proportions of pasture showed significantly reduced mortality when exposed to a lethal concentration of abamectin (0.021 µg/µL) after 48 h. Since abamectin is scarcely used in the study area, we designed an experiment to track ivermectin, a closely related antiparasitic drug used in cattle. Our findings uncovered a new exposure route of bees to pesticides, wherein ivermectin excreted by cattle is absorbed and biotransformed into abamectin within flowering plants in the pastures. These results highlight that unexplained exposure routes of bees to pesticides remain to be described while also revealing that bees adapt to changing landscapes.

Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants.

While genome sequencing has expanded our knowledge of symbiosis, role assignment within multi-species microbiomes remains challenging due to genomic redundancy and the uncertainties of in vivo impacts. We address such questions, here, for a specialized nitrogen (N) recycling microbiome of turtle ants, describing a new genus and species of gut symbiont-Ischyrobacter davidsoniae (Betaproteobacteria: Burkholderiales: Alcaligenaceae)-and its in vivo physiological context. A re-analysis of amplicon sequencing data, with precisely assigned Ischyrobacter reads, revealed a seemingly ubiquitous distribution across the turtle ant genus Cephalotes, suggesting ≥50 million years since domestication. Through new genome sequencing, we also show that divergent I. davidsoniae lineages are conserved in their uricolytic and urea-generating capacities. With phylogenetically refined definitions of Ischyrobacter and separately domesticated Burkholderiales symbionts, our FISH microscopy revealed a distinct niche for I. davidsoniae, with dense populations at the anterior ileum. Being positioned at the site of host N-waste delivery, in vivo metatranscriptomics and metabolomics further implicate I. davidsoniae within a symbiont-autonomous N-recycling pathway. While encoding much of this pathway, I. davidsoniae expressed only a subset of the requisite steps in mature adult workers, including the penultimate step deriving urea from allantoate. The remaining steps were expressed by other specialized gut symbionts. Collectively, this assemblage converts inosine, made from midgut symbionts, into urea and ammonia in the hindgut. With urea supporting host amino acid budgets and cuticle synthesis, and with the ancient nature of other active N-recyclers discovered here, I. davidsoniae emerges as a central player in a conserved and impactful, multipartite symbiosis.

TPGS-750-M: a second-generation amphiphile for metal-catalyzed cross-couplings in water at room temperature.

An environmentally benign surfactant (TPGS-750-M), a diester composed of racemic α-tocopherol, MPEG-750, and succinic acid, has been designed and readily prepared as an effective nanomicelle-forming species for general use in metal-catalyzed cross-coupling reactions in water. Several "name" reactions, including Heck, Suzuki-Miyaura, Sonogashira, and Negishi-like couplings, have been studied using this technology, as have aminations, C-H activations, and olefin metathesis reactions. Physical data in the form of DLS and cryo-TEM measurements suggest that particle size and shape are key elements in achieving high levels of conversion and, hence, good isolated yields of products. This new amphiphile will soon be commercially available.

Assessing Biosynthetic Gene Cluster Diversity of Specialized Metabolites in the Conserved Gut Symbionts of Herbivorous Turtle Ants.

Cephalotes are herbivorous ants (>115 species) feeding on low-nitrogen food sources, and they rely on gut symbionts to supplement their diet by recycling nitrogen food waste into amino acids. These conserved gut symbionts, which encompass five bacterial orders, have been studied previously for their primary nitrogen metabolism; however, little is known about their ability to biosynthesize specialized metabolites which can play a role in bacterial interactions between communities living in close proximity in the gut. To evaluate the biosynthetic potential of their gut symbionts, we mine 14 cultured isolate genomes and gut metagenomes across 17 Cephalotes species to explore the biodiversity of biosynthetic gene clusters (BGCs) producing specialized metabolites. The diversity of BGCs across Cephalotes phylogeny was analyzed using sequence similarity networking and BGC phylogenetic reconstruction. Our results reveal that the conserved gut symbionts involved in the nutritional symbiosis possess 80% of all the 233 BGCs retrieved in this work. Furthermore, the phylogenetic analysis of BGCs reveals different patterns of distribution, suggesting different mechanisms of conservation. A siderophore BGC shows high similarity in a single symbiont across different ant host species, whereas a BGC encoding the production of non-ribosomal peptides (NRPs) found different symbionts within a single host species. Additionally, BGCs were abundant in four of the five bacterial orders of conserved symbionts co-occurring in the hindgut. However, one major symbiont localized alone in the midgut lack BGCs. Because the spatial isolation prevents direct interaction with other symbionts, this result supports the idea that BGCs are maintained in bacteria living in close proximity but are dispensable for an alone-living symbiont. These findings together pave the way for studying the mechanisms of BGC conservation and evolution in gut bacterial genomes associated with Cephalotes. This work also provides a genetic background for further study, aiming to characterize bacterial specialized metabolites and to understand their functional role in multipartite mutualisms between conserved gut symbionts and Cephalotes turtle ants.

Gut bacteria are essential for normal cuticle development in herbivorous turtle ants.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15N isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

UC Pd: a new form of Pd/C for Sonogashira couplings.

Screening of different sources of Pd/C shows reagents of highly variable nanoparticle sizes and oxidation states of the metal. Typically, catalysts with higher surface area are viewed as likely to be the more reactive. In this paper a new form of Pd/C, "UC Pd" is described that is shown to contain larger nanoparticles yet it is the most reactive catalyst of those sold commercially for Sonogashira coupling reactions. UC Pd functions efficiently in the absence of a copper co-catalyst, under very mild and "green" conditions using inexpensive 95% EtOH at 50 degrees C. It is also the only form of Pd/C that can be recycled. In side-by-side reactions with several commercially available forms of Pd/C, none compete successfully with UC Pd under standardized conditions. Physical data obtained from extensive surface analysis using TEM, XRD, XPS, and CO-TPD measurements lead to an explanation behind the unique reactivity of this new recyclable form of Pd/C.

Tracking the Origin and Evolution of Plant Metabolites.

Iridoids are monoterpenes that are produced by various plants as chemical defense molecules. Lichman et al. recently described the timeline of molecular events that underpin the re-emergence of iridoid biosynthesis in an independent lineage of aromatic plants (catnip). This study represents a benchmark for studying enzyme and metabolite evolution in different clades across the tree of life.

Development but not diet alters microbial communities in the Neotropical arboreal trap jaw ant Daceton armigerum: an exploratory study.

To better understand the evolutionary significance of symbiotic interactions in nature, microbiome studies can help to identify the ecological factors that may shape host-associated microbial communities. In this study we explored both 16S and 18S rRNA microbial communities of D. armigerum from both wild caught individuals collected in the Amazon and individuals kept in the laboratory and fed on controlled diets. We also investigated the role of colony, sample type, development and caste on structuring microbial communities. Our bacterial results (16S rRNA) reveal that (1) there are colony level differences between bacterial communities; (2) castes do not structure communities; (3) immature stages (brood) have different bacterial communities than adults; and 4) individuals kept in the laboratory with a restricted diet showed no differences in their bacterial communities from their wild caught nest mates, which could indicate the presence of a stable and persistent resident bacterial community in this host species. The same categories were also tested for microbial eukaryote communities (18S rRNA), and (5) developmental stage has an influence on the diversity recovered; (6) the diversity of taxa recovered has shown this can be an important tool to understand additional aspects of host biology and species interactions.