Chemical Defense Research Articles

Trade-offs among leaf toughness, constitutive chemical defense, and growth rates in oaks are influenced by the level of leaf mass per area

Key messageAmong the variety of leaf defensive strategies to counteract herbivory attacks, the oak species analyzed in this study maximize investment in no more than one, with high-LMA oaks developing very tough leaves and low-LMA oaks favoring between high concentrations of condensed tannins or high growth rates.ContextPlants develop a variety of defense strategies to counteract herbivory attacks, from physical and chemical defenses to tolerance strategies. Tradeoffs between strategies have been widely assessed from a resource allocation perspective, but there is a need to consider eventual interactions among them.AimWe evaluate the among-species tradeoff between three main constitutive plant defense traits, while considering the leaf mass per area ratio (LMA) as a proxy of leaf construction investment on area basis.MethodsLeaf toughness measured as work of fracture, condensed tannins, and relative growth rate (RGR) were analyzed in a set of 19 Quercus L. species with contrasting LMA.ResultsMost species had low values either in two or in the three traits analyzed. Moreover, the highest values of work of fracture appeared in the species with high LMA; the highest values of condensed tannins were found in the species with the lowest LMA; and high values of RGR were measured in species with intermediate or lower values of LMA.ConclusionOaks showed a trade-off among leaf defensive strategies influenced by LMA. Oaks with high LMA developed very tough leaves while oaks with low LMA presented lower values of toughness but favored between one of the two other strategies.

Identification and Localization of Labile Molecules in Broccoli Cell by CLA-ICP-MS and Single-Cell Nanospray HRMS.

Although molecular analysis and imaging by mass spectrometry are emerging as tools to identify metabolites and determine their distribution in cells and tissues, it is difficult to directly analyze the labile molecules at the single-cell level. Glucosinolate (GL) is a plant-active substance with much attention as a chemical defense mechanism known as a "mustard oil bomb" in broccoli. When tissue is damaged, these substances undergo rapid degradation, making them unsuitable for conventional mass spectrometry (MS), particularly for surface MS imaging analysis methods that necessitate intricate preprocessing. Herein, a strategy combining cryogenic laser ablation inductively coupled mass spectrometry (CLA-ICP-MS) and capillary microsampling nanospray high-resolution mass spectrometry (HRMS) was developed. The sulfur-rich microzone in tissue which was thought as a suspect GL-rich cell population was located via CLA-ICP-MS. Three GLs in single cells were accurately identified by nanospray HRMS with a hydrogen/deuterium exchange reaction. Subsequently, cell-by-cell imaging by nanospray HRMS showed that the GL-rich cells were below the stalk surface by approximately 30 μm. This proposed strategy can also be applied to rapidly identify labile compounds and localize molecule-rich cells in tissues.

Evaluating Formic Acid as a Behavioral Modifier in African Savanna Elephants

Formic acid was investigated as a potential repellant for African savanna elephants (Loxodonta africana) as a semiochemical option for managing elephant movements and interactions with human infrastructure. Formic acid is a naturally occurring compound, used as an alarm pheromone and as a defensive chemical in Formicine ants, and thus a potentially desirable option compared to introducing exogenous deterrents that are foreign to the elephants’ natural habitats. Although most elephants observed (85%) did not interact with treatments containing formic acid, of the cohort of individuals (n = 38) that did respond, the majority showed a mild to moderate avoidance response, while a small proportion of elephants were distinctly repelled when experiencing formic acid cues, in some cases causing whole herds to evacuate an area. The potential for using formic acid as an elephant repellant to modulate elephant behavior in field situations is discussed.

Replacing mechanical protection with colorful faces-twice: parallel evolution of the non-operculate marine worm-snail genera Thylacodes (Guettard, 1770) and Cayo n. gen. (Gastropoda: Vermetidae).

Vermetid worm-snails are sessile and irregularly coiled marine mollusks common in warmer nearshore and coral reef environments that are subject to high predation pressures by fish. Often cryptic, some have evolved sturdy shells or long columellar muscles allowing quick withdrawal into better protected parts of the shell tube, and most have variously developed opercula that protect and seal the shell aperture trapdoor-like. Members of Thylacodes (previously: Serpulorbis) lack such opercular protection. Its species often show polychromatic head-foot coloration, and some have aposematic coloration likely directed at fish predators. A new polychromatic species, Thylacodes bermudensis n. sp., is described from Bermuda and compared morphologically and by DNA barcode markers to the likewise polychromatic western Atlantic species T. decussatus (Gmelin, 1791). Operculum loss, previously assumed to be an autapomorphy of Thylacodes, is shown to have occurred convergently in a second clade of the family, for which a new genus Cayo n. gen. and four new western Atlantic species are introduced: C. margarita n. sp. (type species; with type locality in the Florida Keys), C. galbinus n. sp., C. refulgens n. sp., and C. brunneimaculatus n. sp. (the last three with type locality in the Belizean reef) (all new taxa authored by Bieler, Collins, Golding & Rawlings). Cayo n. gen. differs from Thylacodes in morphology (e.g., a protoconch that is wider than tall), behavior (including deep shell entrenchment into the substratum), reproductive biology (fewer egg capsules and eggs per female; an obliquely attached egg capsule stalk), and in some species, a luminous, "neon-like", head-foot coloration. Comparative investigation of the eusperm and parasperm ultrastructure also revealed differences, with a laterally flattened eusperm acrosome observed in two species of Cayo n. gen. and a spiral keel on the eusperm nucleus in one, the latter feature currently unique within the family. A molecular phylogenetic analysis based on mitochondrial and nuclear rRNA gene sequences (12SrRNA, trnV, 16SrRNA, 28SrRNA) strongly supports the independent evolution of the two non-operculate lineages of vermetids. Thylacodes forms a sister grouping to a clade comprising Petaloconchus, Eualetes, and Cupolaconcha, whereas Cayo n. gen is strongly allied with the small-operculate species Vermetus triquetrus and V. bieleri. COI barcode markers provide support for the species-level status of the new taxa. Aspects of predator avoidance/deterrence are discussed for these non-operculate vermetids, which appear to involve warning coloration, aggressive behavior when approached by fish, and deployment of mucous feeding nets that have been shown, for one vermetid in a prior study, to contain bioactive metabolites avoided by fish. As such, non-operculate vermetids show characteristics similar to nudibranch slugs for which the evolution of warning coloration and chemical defenses has been explored previously.

Roles of insect odorant binding proteins in communication and xenobiotic adaptation.

Odorant binding proteins (OBPs) are small water-soluble proteins mainly associated with olfaction, facilitating the transport of odorant molecules to their relevant receptors in the sensillum lymph. While traditionally considered essential for olfaction, recent research has revealed that OBPs are engaged in a diverse range of physiological functions in modulating chemical communication and defense. Over the past 10 years, emerging evidence suggests that OBPs play vital roles in purifying the perireceptor space from unwanted xenobiotics including plant volatiles and pesticides, potentially facilitating xenobiotic adaptation, such as host location, adaptation, and pesticide resistance. This multifunctionality can be attributed, in part, to their structural variability and effectiveness in transporting, sequestering, and concealing numerous hydrophobic molecules. Here, we firstly overviewed the classification and structural properties of OBPs in diverse insect orders. Subsequently, we discussed the myriad of functional roles of insect OBPs in communication and their adaptation to xenobiotics. By synthesizing the current knowledge in this field, our review paper contributes to a comprehensive understanding of the significance of insect OBPs in chemical ecology, xenobiotic adaptation, paving the way for future research in this fascinating area of study.

Selection on Visual Opsin Genes in Diurnal Neotropical Frogs and Loss of the SWS2 Opsin in Poison Frogs.

Amphibians are ideal for studying visual system evolution because their biphasic (aquatic and terrestrial) life history and ecological diversity expose them to a broad range of visual conditions. Here, we evaluate signatures of selection on visual opsin genes across Neotropical anurans and focus on three diurnal clades that are well-known for the concurrence of conspicuous colors and chemical defense (i.e., aposematism): poison frogs (Dendrobatidae), Harlequin toads (Bufonidae: Atelopus), and pumpkin toadlets (Brachycephalidae: Brachycephalus). We found evidence of positive selection on 44 amino acid sites in LWS, SWS1, SWS2, and RH1 opsin genes, of which one in LWS and two in RH1 have been previously identified as spectral tuning sites in other vertebrates. Given that anurans have mostly nocturnal habits, the patterns of selection revealed new sites that might be important in spectral tuning for frogs, potentially for adaptation to diurnal habits and for color-based intraspecific communication. Furthermore, we provide evidence that SWS2, normally expressed in rod cells in frogs and some salamanders, has likely been lost in the ancestor of Dendrobatidae, suggesting that under low-light levels, dendrobatids have inferior wavelength discrimination compared to other frogs. This loss might follow the origin of diurnal activity in dendrobatids and could have implications for their behavior. Our analyses show that assessments of opsin diversification in across taxa could expand our understanding of the role of sensory system evolution in ecological adaptation.

Plant-Plant Interdependence and Mutualism Interactions in Heterogeneous Ecosystems still hide a lot of secrets

Plant-Plant Interdependence and Mutualism Interactions in Heterogeneous Ecosystems still hide a lot of secrets

Plant defences as functional traits: A comparison across savannas differing in herbivore specialization

Abstract Top‐down and bottom‐up forces in tropical savannas have shaped the evolution of traits that enable plants to thrive in environments where soils are poor, dry seasons are long, fire is frequent, woody plants and grasses compete and herbivores range from cryptic caterpillars to elephants that topple entire trees. African savannas and the South American cerrado have similar climates and fire regimes, but differ in the presence of large mammal herbivores, which are mostly extinct in the cerrado. These biomes offer an instructive comparison to understand how plant defence traits evolved in response to differential herbivore pressure. Within Africa, mammalian herbivore pressure also differs across savannas, being lower in the miombo relative to more open, mixed savannas. The dominance of specialist insect herbivores in the cerrado, relative to the more generalist mammalian herbivores in Africa, suggests that defence traits will differ between savannas in accordance with herbivore identity and pressure. Wood is densest in mixed African savannas, as expected because large mammals often damage trees. Specific leaf area, related to palatability, is higher in mixed African savannas and the cerrado relative to the miombo, while leaf N is surprisingly higher in miombo species. Ant mutualists are important defences in mixed African savannas and the cerrado. Defensive chemistry has received less attention in the trait literature. Foliar phenolics, the most commonly studied chemical defences in savannas, vary in their efficacy. New analyses isolating specific compounds are informative as is the evaluation of phytochemical diversity as a defence. There are indications that phytochemical defences are more diverse in the cerrado, which may be related to greater specialization of cerrado herbivores. Synthesis. The dominance of specialist insect herbivores in the cerrado, relative to generalist mammalian herbivores in Africa, selected for different plant defence traits, contributing to the separation of species along the leaf economics spectrum and demonstrating the importance of plant defences as plant functional traits. African savanna species seem to follow a more acquisitive strategy, followed by miombo species. Cerrado species appear to follow a more conservative strategy with greater investments in tough leaves and chemical defences.

Comparing two strategies of counter-defense against plant toxins: a modeling study on plant–herbivore interactions

IntroductionVarious herbivorous insects prefer toxic plants as their hosts, although this may appear paradoxical. They have evolved specific adaptations (called counter-defenses) against the toxins. For example, the two-component chemical defense system of plants of the Brassicaceae family against herbivores consists of glucosinolates (GLSs) and the activating enzyme myrosinase. GLS hydrolysis by myrosinase leads to isothiocyanates (ITCs), which are toxic and deterrent to many insect herbivores. Two different types of counter-defenses can be distinguished: a preemptive counter-defense that prevents the GLSs from being hydrolyzed to ITCs due to metabolic redirection and direct counter-defense, where the ITCs are formed but then metabolized to non-toxic conjugates. In general, preemptive counter-defense is only a possibility if the plant stores a precursor of the toxin, which is activated upon attack. Preemptive counter-defense is believed to be more efficient due to the lower exposure to ITCs, but this has not been satisfactorily demonstrated experimentally.MethodsHere, we derive on theoretical grounds the conditions under which preemptive counter-defense reduces exposure to ITCs compared to direct counter-defense by studying the dynamics of GLS defense and counter-defense with two separate ordinary differential equation models. We model how herbivory transfers the GLSs to the insect gut with the leaf material. Thereafter, we describe the insects’ exposure to toxins by deriving the dynamics of ITCs in the gut during feeding with the two types of counter-defenses.Results and discussionBy calculating the area under the curve (AUC) of the ITC concentrations, we show, based on empirical data, that herbivores with a preemptive detoxification system are usually less exposed to ITCs. In addition, our models explain how the decline in the level of ITCs is achieved by both counter-defenses, which helps to understand the overall mechanisms and benefits of these techniques. Our results may also apply to plant defenses by inactive toxin precursors other than GLSs as well as to insects that sequester such precursors for their own defense.

Efficacy of a monovalent (D614) SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant in adults: a phase 3, multi-country study

The literature on first generation COVID-19 vaccines show they were less effective against new SARS-CoV-2 variants of concern including Omicron (BA.1, BA.2, BA.4 and BA.5 subvariants). New vaccines developed against variant strains may provide cross-protection against emerging variants when used as boosters and facilitate vaccination across a range of countries, healthcare settings and populations. However, there are no data on such vaccines when used as a primary series. A global Phase 3, multi-stage efficacy study (NCT04904549) among adults (≥18 years) was conducted in 53 research centres in eight countries (United States, Honduras, Japan, Colombia, Kenya, India, Ghana, Nepal). Participants were randomized 1:1 to receive two intramuscular injections of a monovalent SARS-CoV-2 recombinant protein vaccine with AS03-adjuvant (10μg of the spike (S) protein from the ancestral D614 strain) or placebo on Day 1 (D01) and Day 22 (D22). The primary efficacy endpoint was prevention of virologically confirmed SARS-CoV-2 infection with symptoms of COVID-19-like illness (CLI) ≥14 days after the second injection (post-dose 2 [PD2]) in participants who were SARS-CoV-2 naïve on D01+D22. Safety and reactogenicity were also evaluated. Between May 26 and November 7, 2021, 10,114 participants received ≥1 study injection, and 9441 participants received both injections. 2108 (20.8%) participants were SARS-CoV-2 naïve at D01 and D22. The primary endpoint was analysed in a subset of the full analysis set (the modified full analysis set PD2 [mFAS-PD2], excluding participants who did not complete the vaccination schedule or received vaccination despite meeting one of the contraindication criteria, had onset of symptomatic COVID-19 between the first injection and before 14 days after the second injection, or participants who discontinued before 14 days after the second injection [n=9377; vaccine, n=4702; placebo, n=4675]). Data were available for 2051 SARS-CoV-2 naïve and 7159 non-naïve participants. At the cut-off date (January 28, 2022), symptomatic COVID-19 was reported in 169 naïve participants (vaccine, n=81; placebo, n=88) ≥14 days PD2, with a vaccine efficacy (VE) of 15.3% (95% CI,-15.8; 38.2). VE regardless of D01/D22 serostatus was 32.9% (95% CI, 15.3; 47.0) and VE in non-naïve participants was 52.7% (95% CI, 31.2; 67.9). Viral genome sequencing was performed up to the data cut-off point and identified the infecting strain in 99/169 adjudicated cases in the PD2 naïve population (Delta [25], Omicron [72], other variants [3], one participant had infection with both Delta and Omicron variants and has been included in the totals for both Delta and Omicron). The vaccine was well-tolerated with an acceptable safety profile. In the context of changing circulating viral variants, it is challenging to induce protection in naïve individuals with a two-dose priming schedule based on the parental D614 strain. However, while the primary endpoint of this trial was not met, the results show that a monovalent D614 vaccine can still be of value in individuals previously exposed to SARS-CoV-2. This study was funded in whole or in part by Sanofi and by federal funds from the Biomedical Advanced Research and Development Authority, part of the office of the Administration for Strategic Preparedness and Response at the U.S. Department of Health and Human Services under contract number HHSO100201600005I, and in collaboration with the U.S. Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense under contract number W15QKN-16-9-1002. The views presented here are those of the authors and do not purport to represent those of the Department of the Army, the Department of Health and Human Services, or the U.S. government.

Chemical analysis of endophytic fungi isolated from mangrove trees in Playa San Pedro Nature Reserve, Buenaventura, Valle del Cauca, Colombia

Endophytic fungi are well known for their association with a wide variety of plant species, likewise, mangrove plants are well known for harboring a vast variety of fungi with a valuable diversity of bioactive compounds originating from the secondary metabolism that is synthesized in part as a response to the chemical defense against microorganisms, hostile environments, and antagonistic insects. The objective of the present study was to analyze the chemical composition of endophytic fungi isolated from mangrove trees in Buenaventura, Colombia. Analyses of DNA sequences from the internal transcribed spacer ribosomal nuclear region (ITS) were conducted to determine the fungi’s identity. The results revealed 17 isolates, belonging to eight fungal families. All isolates were subjected to thin-layer chromatography analysis, observing different phytochemical nuclei eluted in the system (7: 3 hexane: acetone), of these, 23 compounds were recognized using gas chromatography coupled to mass spectrometry; cytotoxicity tests were carried out in human foreskin fibroblast cell line, which did not show a trend in cell viability. The selected endophytic fungi derived from mangrove trees reveal the presence of different chemical compounds, representing an alternative resource of great interest in bioprospecting and bioremediation.

Temperature alters the toxicological impacts of plant terpenoids on the polyphagous model herbivore Vanessa cardui

Terpenes are a major class of secondary metabolites present in all plants, and long hypothesized to have diversified in response to specific plant-herbivore interactions. Herbivory is a major biotic interaction that plays out across broad temporal and spatial scales that vary dramatically in temperature regimes, both due to climatic variation across geographic locations as well as the effect of seasonality. In addition, there is an emerging understanding that global climate change will continue to alter the temperature regimes of nearly every habitat on Earth over the coming centuries. Regardless of source, variation in temperature may influence herbivory, in particular via changes in the efficacy and impacts of plant defensive chemistry. This study aims to characterize temperature-driven variation in toxicological effects across several structural classes of terpenes in the model herbivore Vanessa cardui, the painted lady butterfly. We observed a general increase in monoterpene toxicity to larvae, pupa, and adults at higher temperatures, as well as an increase in development time as terpene concentration increased. Results obtained from this study yield insights into possible drivers of seasonal variation in plant terpene production as well as inform effects of rising global temperatures on plant-insect interactions. In the context of other known effects of climate change on plant-herbivore interactions like carbon fertilization and compensatory feeding, temperature-driven changes in plant chemical defense efficacy may further complicate the prediction of climate change impacts on the fundamental ecological process of herbivory.

Long term effects of ionising radiation in the Chernobyl Exclusion zone on DNA integrity and chemical defence systems of Scots pine (Pinus sylvestris)

The Chernobyl Nuclear Power Plant (ChNPP) accident in 1986 resulted in extremely high levels of acute ionising radiation, that killed or damaged Scots pine (Pinus sylvestris) trees in the surrounding areas. Dead trees were cleared and buried, and new plantations established a few years later. Today, more than three decades later, gamma and beta-radiation near the ChNPP is still elevated compared with ambient levels but have decreased by a factor of 300 and 100, respectively. In the present work, Scots pine-trees growing at High (220 μGy h−1), Medium (11 μGy h−1), and Low (0.2 μGy h−1) total (internal + external) dose rates of chronically elevated ionising radiation in the Chernobyl Exclusion zone were investigated with respect to possible damage to DNA, cells and organelles, as well as potentially increased levels of phenolic and terpenoid antioxidants. Scots pine from the High and Medium radiation sites had elevated levels of DNA damage in shoot tips and needles as shown by the COMET assay, as well as increased numbers of resin ducts and subcellular abnormalities in needles. Needles from the High radiation site showed elevated levels of monoterpenes and condensed tannins compared with those from the other sites. In conclusion, more than three decades after the ChNPP accident substantial DNA damage and (sub)cellular effects, but also mobilisation of stress-protective substances possessing antioxidant activity were observed in Scots pine trees growing at elevated levels of ionising radiation. This demonstrates that the radiation levels in the Red Forest still significantly impact the plant community.

The effects of P-glycoprotein induction on ivermectin-induced behavioural alterations in zebrafish (Danio rerio) under varying diets

The neuroprotective effects of inducing the blood-brain barrier ATP-binding cassette protein transporter P-glycoprotein (P-gp) with clotrimazole (CTZ) in both fed and fasted zebrafish (Danio rerio) against the CNS-toxicant ivermectin (IVM, 22,23-dihydro avermectin B1a + 22,23-dihydro avermectin B1b) were examined. Zebrafish were administered 2 μmol/kg IVM intraperitoneally, and various behavioural assays (swimming performance, exploratory behaviour, olfactory responses, motor coordination, and escape responses) were used to measure neurological dysfunction. IVM administration alone caused a decrease in mean swim speed (91 % of controls), maximal speed (71 %), passage rate (81 %), 90° turns (81 %), and response to food stimulus (39 %). IVM exposure also increased the percent time that fish spent immobile (45 % increase over controls) and the percent of lethargic fish (40 % increase). Fish administered 30 μmol/kg of the P-gp inducer CTZ intraperitoneally 3 d prior to IVM exposure exhibited a change in only the % time spent immobile. These data indicate that P-gp induction may be limited in protecting the zebrafish CNS from IVM over baseline. Fasted fish did not differ from fed fish in the effects of IVM on behaviour, and no differences were seen following P-gp induction with CTZ. These results suggest that this chemical defence system is not downregulated when fish are challenged with limited energy availability.

Larvae of Sasakia charonda (Lepidoptera: Nymphalidae: Apaturinae) and three related species use oral odorants to repel ants and wasps

We incidentally discovered that the larvae of Sasakia charonda (Hewitson, 1863) (Lepidoptera: Nymphalidae: Apaturinae) disturbed by ants, wasps, or humans release volatile compounds orally. To identify these substances, we collected oral odorant samples directly from the mouths of S. charonda larvae into volatile-collecting tubes. The trapped oral odorant samples were subjected to gas chromatography–mass spectrometry (GC–MS). We confirmed the identity of 19 substances by comparing them to GC results of known standards and inferred them to mainly be alcohols and aldehydes/ketones, with main chains of 4–5 carbons. Three of the chemicals in the oral odorant samples, 2-butanol, 1-penten-3-ol, and 3-pentanone, showed a repellant effect on the ants Pristomyrmex punctatus (Smith, 1860) and Formica japonica Motschoulsky, 1866 (Hymenoptera: Formicidae). We also examined the effects of these 19 volatiles on Polistes spp. (Hymenoptera: Vespidae) and confirmed that some of them were effective repellants against these wasps. We propose that the substances we identified in this study can be used as defensive chemicals, analogous to the osmeterium emissions specific to Papilionidae butterflies. Furthermore, we examined the oral odorants of three related Japanese Apaturine species, Hestina assimilis (Linnaeus, 1758), H. persimilis (Westwood, 1850), and Apatura metis (Freyer, 1829) using the same approach. The chemical compositions of the oral odorants of H. assimilis and H. persimilis were similar to that of S. charonda, whereas that of A. metis differed. Some of the oral substances also induced a defensive response in conspecific Apaturinae larvae. We consider these substances to also act as alarm pheromones in these larvae.

Leaf metabolic traits reveal hidden dimensions of plant form and function.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization-a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.

Evolutionary host shifts across plant orders despite high host specificity in tree stem surface-living Stomaphis aphids inferred from molecular phylogeny

We reconstructed Stomaphis phylogeny and analyzed evolutionary host-plant shifts. The molecular phylogeny revealed 23 well-supported lineages, each specialized to use specific host plant family, whilst host plants of Stomaphis aphids ranged across 6 orders, 11 families, 21 genera, and 28 species. This combination of high host specificity with evolutionarily distant host shifts is exceptional in herbivores. To explain this pattern, we propose one hypothesis among several possibilities: Stomaphis aphids are generalists with respect to the defensive chemicals produced by the plant, but specialists with respect to the stem surface structure of the host tree. This hypothesis predicts that tree taxa having stem surface structures preferred by Stomaphis would be used again and again by genetically distinct Stomaphis lineages. Consistent with this prediction, we found that different (occasionally phylogenetically distant) Stomaphis lineages shared the same host plant genera such as Alnus and Betula. This result suggests that, in the course of their evolutionary history, Japanese Stomaphis aphids have repeatedly colonized a limited number of host plant genera.

A test of the Geographic Mosaic Theory of Coevolution: investigating widespread species of Amazonian Protium (Burseraceae) trees, their chemical defenses, and their associated herbivore faunas

IntroductionPlants and their insect herbivores represent a large fraction of the species in Amazonian forests and are often directly implicated in the origin and maintenance of biodiversity at local and regional scales. How these interactions may change over geographic distance is unknown because very few studies have investigated the herbivore fauna and defense chemicals of any host plant species at multiple sites in tropical forests. One hypothesis, the Geographic Mosaic Theory of Coevolution, predicts that if herbivore assemblages turn over in different parts of a plant’s range, then plant defense chemicals should also change, reflecting local selection pressures.MethodsWe tested this theory by studying 12 species of Protium (Burseraceae) trees that occur in both Iquitos, Peru, and Manaus, Brazil, in rainforests separated by 1500 km. We surveyed all insects observed directly feeding on the plants in both locations for 48 weeks in Manaus and 64 weeks in Iquitos. We analyzed the secondary metabolites in the leaves of all species in both locations using GC/MS and HPLC.Results and DiscussionAlthough in both locations we found that Protium herbivores were dominated by insects from the orders Hemiptera, Coleoptera and Lepidoptera, we found almost complete turnover in the herbivore species composition in the two sites, and each host plant species had a different assemblage of herbivores in each location. Comparing the phylogenetic beta-diversity, we found low similarity in herbivore phylogenetic relatedness between host plant species in the two locations. However, the secondary metabolites found within a Protium species were similar across the two locations. We found no strong evidence that individuals from a host plant species in Iquitos or Manaus expressed locally-adapted defense chemicals, as individuals from geographic locations did not form clusters when looking at patterns of chemical similarity. These results are not consistent with the Geographic Mosaic Theory of Coevolution. The most intriguing pattern we found was a strong correlation between the diversity of herbivores per host plant species in both locations. We also found that plants with high chemical richness had lower numbers of herbivore species and numbers of total herbivores in both locations. We conclude that high chemical diversity is the most effective strategy for Protium trees to reduce insect herbivore attacks. We speculate that each secondary metabolite is effective at repelling only a few insect herbivores, and that different chemicals are likely effective in different parts of a plants’ geographic range. Future studies should investigate additional locations and additional natural enemies (i.e., fungal pathogens) to test the hypothesis that chemical diversity reduces attack from natural enemies and may explain the ecological and evolutionary success of rainforest trees over time and space.

Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopy.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Functional characterization and structural basis of a reversible glycosyltransferase involves in plant chemical defence.

Plants experience numerous biotic stresses throughout their lifespan, such as pathogens and pests, which can substantially affect crop production. In response, plants have evolved various metabolites that help them withstand these stresses. Here, we show that two specialized metabolites in the herbaceous perennial Belamcanda chinensis, tectorigenin and its glycoside tectoridin, have diverse defensive effects against phytopathogenic microorganisms and antifeeding effects against insect pest. We further functionally characterized a 7-O-uridine diphosphate glycosyltransferase Bc7OUGT, which catalyses a novel reversible glycosylation of tectorigenin and tectoridin. To elucidate the catalytic mechanisms of Bc7OUGT, we solved its crystal structure in complex with UDP and UDP/tectorigenin respectively. Structural analysis revealed the Bc7OUGT possesses a narrow but novel substrate-binding pocket made up by plentiful aromatic residues. Further structure-guided mutagenesis of these residues increased both glycosylation and deglycosylation activities. The catalytic reversibility of Bc7OUGT was also successfully applied in an one-pot aglycon exchange reaction. Our findings demonstrated the promising biopesticide activity of tectorigenin and its glycosides, and the characterization and mechanistic study of Bc7OUGT could facilitate the design of novel reversible UGTs to produce valuable glycosides with health benefits for both plants and humans.