Chemical Defense Research Articles

Genome-Wide Analysis and Expression Profiling of the Glutathione Peroxidase-like Enzyme Gene Family in Solanum tuberosum.

Glutathione peroxidase-like enzyme is an important enzymatic antioxidant in plants. It is involved in scavenging reactive oxygen species, which can effectively prevent oxidative damage and improve resistance. GPXL has been studied in many plants but has not been reported in potatoes, the world's fourth-largest food crop. This study identified eight StGPXL genes in potatoes for the first time through genome-wide bioinformatics analysis and further studied the expression patterns of these genes using qRT-PCR. The results showed that the expression of StGPXL1 was significantly upregulated under high-temperature stress, indicating its involvement in potato defense against high-temperature stress, while the expression levels of StGPXL4 and StGPXL5 were significantly downregulated. The expression of StGPXL1, StGPXL2, StGPXL3, and StGPXL6 was significantly upregulated under drought stress, indicating their involvement in potato defense against drought stress. After MeJA hormone treatment, the expression level of StGPXL6 was significantly upregulated, indicating its involvement in the chemical defense mechanism of potatoes. The expression of all StGPXL genes is inhibited under biotic stress, which indicates that GPXL is a multifunctional gene family, which may endow plants with resistance to various stresses. This study will help deepen the understanding of the function of the potato GPXL gene family, provide comprehensive information for the further analysis of the molecular function of the potato GPXL gene family as well as a theoretical basis for potato molecular breeding.

Not just the sum of its parts: Geographic variation and nonadditive effects of pyrazines in the chemical defence of an aposematic moth.

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within- and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine), are volatiles that deter bird attacks. We hypothesized that (1) variation in the chemical defences of male wood tiger moths reflects the local predation pressure; (2) observed differences in quantity and quality of defence among populations have a genetic basis; and (3) increasing concentrations of SBMP and IBMP will elicit greater aversive reactions in predators, with the two pyrazines having an additive effect on predators' avoidance. We found that (1) the chemical defence of wild moths partly reflects local predator selection: high predation pressure populations (Scotland and Georgia) had stronger chemical defences, but not lower variance, than the low-predation populations (Estonia and Finland). (2) Based on the common garden results, both genetic and environmental components seem to influence the strength of chemical defence in moth populations; and (3) IBMP alone did not provide protection against bird predators but worked against bird attacks only when combined with SBMP, and while SBMP was more effective at higher concentrations, IBMP was not. Altogether this suggests that, when it comes to pyrazine concentration, more is not always better, highlighting the importance of testing the efficacy of chemical defence and its components with relevant predators, as extrapolating from chemical data may be less than straightforward.

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs.

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa - spiders and birds - to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.

The protective value of the colour and shape of the mountain katydid's antipredator defence.

Deimatic behaviour is performed by prey when attacked by predators as part of an antipredator strategy. The behaviour is part of a sequence that consists of several defences, for example they can be preceded by camouflage and followed by a hidden putatively aposematic signal that is only revealed when the deimatic behaviour is performed. When displaying their hidden signal, mountain katydids (Acripeza reticulata) hold their wings vertically, exposing striking red and black stripes with blue spots and oozing an alkaloid-rich chemical defence derived from its Senecio diet. Understanding differences and interactions between deimatism and aposematism has proven problematic, so in this study we isolated the putative aposematic signal of the mountain katydid's antipredator strategy to measure its survival value in the absence of their deimatic behaviour. We manipulated two aspects of the mountain katydid's signal, colour pattern and whole body shape during display. We deployed five kinds of clay models, one negative control and four katydid-like treatments, in 15 grids across part of the mountain katydid's distribution to test the hypothesis that their hidden signal is aposematic. If this hypothesis holds true, we expected that the models, which most closely resembled real katydids would be attacked the least. Instead, we found that models that most closely resembled real katydids were the most likely to be attacked. We suggest several ideas to explain these results, including that the deimatic phase of the katydid's display, the change from a camouflaged state to exposing its hidden signal, may have important protective value.

Quantitative Chemical Analysis of Defensive Secretion of Megacrania tsudai (Phasmatidae) and Effect of Actinidine on its Potential Predators.

Insects avoid predation in various ways, and some use multiple ways to avoid predation. However, the effects of comprehensive avoidance methods and the differences in avoidance methods among different life stages of insects have not been sufficiently discussed. The big head stick insect Megacrania tsudai uses background matching as its primary defense and chemical defense as its secondary defense. The aims of this study were to identify and isolate the chemical components of M. tsudai using repeatable methods, determine the amount of the main chemical compound, and reveal the effect of the main chemical compound on its predators. We established a repeatable gas chromatography-mass spectrometry (GC-MS) method to identify the chemical compounds of these secretions, and identified actinidine as the main compound. Actinidine was identified by nuclear magnetic resonance (NMR), and the amount of actinidine in each instar was calculated by constructing a calibration curve using pure actinidine. Mass ratios did not drastically change among instars. Furthermore, experiments involving dropping an aqueous solution of actinidine demonstrated removal behavior in geckos, frogs, and spiders. These results indicated that M. tsudai conducts secondary defenses using defensive secretions consisting mainly of actinidine.

Chemical profile and analysis of biosynthetic pathways and genes of volatile terpenes in Pityopsis ruthii, a rare and endangered flowering plant.

It is critical to gather biological information about rare and endangered plants to incorporate into conservation efforts. The secondary metabolism of Pityopsis ruthii, an endangered flowering plant that only occurs along limited sections of two rivers (Ocoee and Hiwassee) in Tennessee, USA was studied. Our long-term goal is to understand the mechanisms behind P. ruthii's adaptation to restricted areas in Tennessee. Here, we profiled the secondary metabolites, specifically in flowers, with a focus on terpenes, aiming to uncover the genomic and molecular basis of terpene biosynthesis in P. ruthii flowers using transcriptomic and biochemical approaches. By comparative profiling of the nonpolar portion of metabolites from various tissues, P. ruthii flowers were rich in terpenes, which included 4 monoterpenes and 10 sesquiterpenes. These terpenes were emitted from flowers as volatiles with monoterpenes and sesquiterpenes accounting for almost 68% and 32% of total emission of terpenes, respectively. These findings suggested that floral terpenes play important roles for the biology and adaptation of P. ruthii to its limited range. To investigate the biosynthesis of floral terpenes, transcriptome data for flowers were produced and analyzed. Genes involved in the terpene biosynthetic pathway were identified and their relative expressions determined. Using this approach, 67 putative terpene synthase (TPS) contigs were detected. TPSs in general are critical for terpene biosynthesis. Seven full-length TPS genes encoding putative monoterpene and sesquiterpene synthases were cloned and functionally characterized. Three catalyzed the biosynthesis of sesquiterpenes and four catalyzed the biosynthesis of monoterpenes. In conclusion, P. ruthii plants employ multiple TPS genes for the biosynthesis of a mixture of floral monoterpenes and sesquiterpenes, which probably play roles in chemical defense and attracting insect pollinators alike.

Integration of transcriptomic and metabolomic analysis unveils the response mechanism of sugar metabolism in Cyclocarya paliurus seedlings subjected to PEG-induced drought stress

Cyclocarya paliurus (Batal.) Iljinskaja is a multiple function tree species used for functional food and valued timber production. Carbohydrates, especially water-soluble carbohydrates, play an important role in osmotic protection, signal transduction and carbon storage. Under the circumstance of global climate change the abiotic stress would restrict the development of C. paliurus plantation, whereas there is few knowledge on the regulatory mechanisms of sugar metabolism under drought stress in C. paliurus. To investigate the drought response of C. paliurus at molecular level, we conducted an integrated analysis of transcriptomic and metabolomic of C. paliurus at three PEG-induced drought stress levels (0%: control; 15%: moderate drought; 25%: severe drought) in short term. Both moderate and severe drought treatments activated the chemical defense with lowering relative water content, and enhancing the contents of soluble protein, proline and malondialdehyde in the leaves. Meanwhile, alterations in the expression of differentially expressed genes and carbohydrate metabolism profiles were observed among the treatments. Weighted gene co-expression network analysis (WGCNA) showed 3 key modules, 8 structural genes (such as genes encoding beta-fructofuranosidase (INV), sucrose synthase (SUS), raffinose synthase (RS)) and 14 regulatory transcription factors were closely linked to sugar metabolism. Our results provided the foundation to understand the response mechanism of sugar metabolism in C. paliurus under drought stress, and would drive progress in breeding of drought-tolerant varieties and plantation development of the species.

Proteins from toad’s parotoid macroglands: do they play a role in gland functioning and chemical defence?

BackgroundParotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands.ResultsApplying a proteomic approach we identified 162 proteins in the extract from toad’s parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings.ConclusionThis finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

The effects of chilling and forcing temperatures on spring synchrony between larch casebearer and tamarack

Abstract Spring phenological synchrony can be important for tree‐insect interactions. Depending on the magnitude and direction of phenological shifts, overwintering insects could be affected in many ways, for example, facing starvation or having to contend with increased chemical or physical defences of host trees. If temperature has different influences on the phenology of trees and insects, climate change can alter spring phenological synchrony. In this experiment, we exposed tamarack seedlings and larch case bearer larvae from Minnesota, USA, to a variety of chilling and forcing temperatures and measured spring phenology (twig bud break and larval activation). We additionally measured case bearer performance on seedlings that were exposed to different forcing × chilling levels, tracking larval survivorship to adulthood. Warmer forcing enhanced larval activation and bud break, but larval development slowed down past 21°C. Higher chilling temperatures accelerated bud break, but the effect was inconclusive for larvae. There was no chilling × forcing interaction for either species. Spring activity accelerated more quickly with increases in temperature for larvae than for seedlings, resulting in increased phenological synchrony at warmer temperatures. Activation rates for overwintering larvae were highest at 27°C, while survivorship to adulthood following spring activation was highest at 21°C. At temperatures at or beyond 27°C, no larvae reached adulthood. Warmer winters and springs will likely initially increase spring synchrony between tamarack and larch case bearer, exposing larvae to younger, potentially more nutritious foliage, but extremely warm spring temperatures may decrease survivorship of larvae to adulthood.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

BackgroundNudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.ResultsWe recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously.ConclusionsAltogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.8entVVL2Hhkfc-ejj4sDuQVideo

Coevolutionary escalation led to differentially adapted paralogs of an insect's Na,K-ATPase optimizing resistance to host plant toxins.

Cardiac glycosides are chemical defence toxins known to fatally inhibit the Na,K-ATPase (NKA) throughout the animal kingdom. Several animals, however, have evolved target-site insensitivity through substitutions in the otherwise highly conserved cardiac glycoside binding pocket of the NKA. The large milkweed bug, Oncopeltus fasciatus, shares a long evolutionary history with cardiac glycoside containing plants that led to intricate adaptations. Most strikingly, several duplications of the bugs' NKA1α gene provided the opportunity for differential resistance-conferring substitutions and subsequent sub-functionalization of the enzymes. Here, we analysed cardiac glycoside resistance and ion pumping activity of nine functional NKA α/β-combinations of O. fasciatus expressed in cell culture. We tested the enzymes with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a standard cardiac glycoside. The identity and number of known resistance-conferring substitutions in the cardiac glycoside binding site significantly impacted activity and toxin resistance in the three α-subunits. The β-subunits also influenced the enzymes' characteristics, yet to a lesser extent. Enzymes containing the more ancient αC-subunit were inhibited by both compounds but much more strongly by the host plant toxin calotropin than by ouabain. The sensitivity to calotropin was diminished in enzymes containing the more derived αB and αA, which were only marginally inhibited by both cardiac glycosides. This trend culminated in αAβ1 having higher resistance against calotropin than against ouabain. These results support the coevolutionary escalation of plant defences and herbivore tolerance mechanisms. The possession of multiple paralogs additionally mitigates pleiotropic effects by compromising between ion pumping activity and resistance.

Differential contributions of two domains of NAI2 to the formation of the endoplasmic reticulum body.

The endoplasmic reticulum (ER) serves essential functions in eukaryotic cells, including protein folding, transport of secretory proteins, and lipid synthesis. The ER is a highly dynamic organelle that generates various types of compartments. Among them, the ER body is specifically present in plants in the Brassicaceae family and plays a crucial role in chemical defense against pathogens. The NAI2 protein is essential for ER body formation, and its ectopic overexpression is sufficient to induce ER body formation even in the leaves of Nicotiana benthamiana, where the ER body does not naturally exist. Despite the significance of NAI2 in ER body formation, the mechanism whereby NAI2 mediates ER body formation is not fully clear. This study aimed to investigate how two domains of Arabidopsis NAI2, the Glu-Phe-Glu (EFE) domain (ED) and the NAI2 domain (ND), contribute to ER body formation in N. benthamiana leaves. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we found that the ND is critical for homomeric interaction of NAI2 and ER body formation. Moreover, deletion of ED induced the formation of enlarged ER bodies, suggesting that ED plays a regulatory role during ER body formation. Our results indicate that the two domains of NAI2 cooperate to induce ER body formation in a balanced manner.

Integrating defense and leaf economic spectrum traits in a tropical savanna plant.

Allocation to plant defense traits likely depends on resource supply, herbivory, and other plant functional traits such as the leaf economic spectrum (LES) traits. Yet, attempts to integrate defense and resource acquisitive traits remain elusive. We assessed intraspecific covariation between different defense and LES traits in a widely distributed tropical savanna herb, Solanum incanum, a unique model species for studying allocations to physical, chemical, and structural defenses to mammalian herbivory. We found that in a multivariate trait space, the structural defenses - lignin and cellulose - were positively related to the resource conservative traits - low SLA and low leaf N. Phenolic content, a chemical defense, was positively associated with resource acquisitive traits - high SLA and high leaf N - while also being associated with an independent third component axis. Both principal components 1 and 3 were not associated with resource supply and herbivory intensity. In contrast, spine density - a physical defense - was orthogonal to the LES axis and positively associated with soil P and herbivory intensity. These results suggest a hypothesized "pyramid" of trade-offs in allocation to defense along the LES and herbivory intensity axes. Therefore, future attempts to integrate defense traits with the broader plant functional trait framework, such as the LES, needs a multifaceted approach that accounts for unique influences of resource acquisitive traits and herbivory risk.

The effects of plants domestication on the foraging and performance of parasitoids.

Domestication-related changes in the chemical traits of crop plants affect parasitoid foraging success, development, and survival. For example, herbivore-induced changes in the production of volatiles by domesticated plants can enhance or reduce parasitoid attraction. While the trade-off between nutrient content and chemical defense in cultivated plants can increase the suitability of hosts for parasitoids, their increased health and size can positively affect their immune response against parasitoids. Overall, plant domestication is expected to significantly affect their relationship with parasitoids due to altered plant morphology, physical characteristics, chemical defenses, and new plant associations. This review highlights the need for research on the effects of plant domestication on host-parasitoid interactions in the interest of better controlling insect pests.

Why are phytoseiid predatory mites not effectively controlling Echinothrips americanus?

The poinsettia thrips, Echinothrips americanus Morgan (Thysanoptera: Thripidae), is a key pest of various ornamental and vegetable greenhouse crops. As current biological control alternatives lack efficiency, applying chemicals remains the dominant control strategy, thereby heavily disturbing the biocontrol-based integrated management of other pests. For a range of other thrips pests, phytoseiid predatory mites have shown to be effective biocontrol agents, being able to overcome the thrips' physical and chemical defense armory. Here, we investigated potential underlying causes for the lack of phytoseiid efficacy in controlling E. americanus. First, we assessed the nutritional value of E. americanus for the predatory mite Amblydromalus limonicus (Garman and McGregor) (Acari: Phytoseiidae) when its physical or chemical defenses were eliminated by freezing the thrips. The phytoseiid could complete its immature development when frozen thrips instars were offered, but not when these were offered alive. Subsequently, we tested whether adult female A. limonicus had a higher predation rate on first instar E. americanus when they had been given experience with either live or frozen E. americanus during their immature development (i.e., conditioning). Conditioning significantly increased the predation capacity of the phytoseiid. Finally, we tested the control potential of conditioned A. limonicus versus naïve ones when exposed to E. americanus on sweet pepper plants. In contrast to the laboratory trials, at the plant level, conditioning did not yield better control. Possible factors explaining insufficient control of E. americanus by phytoseiids are discussed.

Evidence for some antimicrobial properties of English churchyard lichens.

The emergence of multidrug-resistant bacteria has driven the need for novel antibiotics. Our investigations have focussed on lichens as they naturally produce a wide range of unique and very effective defence chemicals. The aim of this study was to evaluate some of the antimicrobial properties of ten common British churchyard lichens. The lichen material was sampled from ten species, namely Caloplaca flavescens, Diploicia canescens, Cladonia fimbriata, Psilolechia lucida, Lecanora campestris subsp. Campestris, Lecanora sulphurea, Pertusaria amara f.amara, Lepraria incana, Porpidia tuberculosa and Xanthoria calcicola. Crude acetone extracts of these lichens were tested against six bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonela typhimurium, Listeria monocytogenes and Lactobacillus acidophilus ) and two fungi (Trichophyton interdigitale and Aspergillus flavus) by the disc-diffusion susceptibility test method. Extracts of Diploicia canescens, Psilolechia lucida, Lecanora sulphurea, Pertusaria amara and Lepraria incana showed clear inhibition of the Gram-positive bacteria tested (S. aureus, L. monocytogenes, L. plantarum). Diploicia canescens, Pertusaria amara and Lepraria incana extracts also inhibited the dermatophyte fungi tested. The Lepraria incana sample tested here was the only extract that showed activity against any of the Gram-negative bacteria tested; it showed inhibition of Pseudomnas aeruginosa. Overall, our results showed that crude extracts of Diploicia canescens and Pertusaria amara had the most potent antimicrobial activity of all the extracts tested. Our results are in general agreement with published findings elsewhere. The activity of the Porpidia tuberculosa margin sample being different from that of the main colony material was an interesting and new finding reported here for the first time.

Asymmetric interactions between two butterfly species mediated by food demand.

Recent studies on insect interactions on plants have revealed that herbivorous insects indirectly interact with each other through changes in plant traits following herbivory. However, less attention has been given to plant biomass relative to plant quality in relation to indirect interactions among herbivores. We explored the extent to which the larval food demand of two specialist butterflies (Sericinus montela and Atrophaneura alcinous) explains their interaction on a host plant, Aristolochia debilis. A laboratory experiment showed that plant mass consumption by A. alcinous larvae was 2.6 times greater than that by S. montela. We predicted that A. alcinous, which requires more food, is more vulnerable to food shortages than S. montela. In a cage experiment, an asymmetric interspecific interaction was detected between the two specialist butterflies; S. montela larval density significantly decreased the survival and prolonged the development time of A. alcinous, but A. alcinous density affected neither the survival nor the development time of S. montela. The prediction based on the food requirement was partly supported by the fact that increasing A. alcinous density likely caused a food shortage, which more negatively affected A. alcinous survival than S. montela survival. Conversely, increasing the density of S. montela did not reduce the remaining food quantity, suggesting that the negative effect of S. montela density on A. alcinous was unlikely to be due to food shortage. Although aristolochic acid I, a defensive chemical specific to Aristolochia plants, did not influence the food consumption or growth of either butterfly larva, unmeasured attributes of plant quality may have mediated an indirect interaction between the two butterflies. Consequently, our study suggests that not only the quality but also the quantity of plants should be considered to fully understand the characteristics, such as symmetry, of interspecific interactions among herbivorous insects on the same host plant.

Effects of air humidity and soil moisture on secondary metabolites in the leaves and roots of Betula pendula of different competitive status.

Plant secondary metabolites (PSMs) defend plants against abiotic stresses, including those caused by climate change and against biotic stresses, such as herbivory and competition. There is a trade-off between allocating available carbon to growth and defence in stressful environments. However, our knowledge about trade-off is limited, especially when abiotic and biotic stresses co-occur. We aimed to understand the combined effect of increasing precipitation and humidity, the tree's competitive status, and canopy position on leaf secondary metabolites (LSMs) and fine root secondary metabolites (RSMs) in Betula pendula. We sampled 8-year-old B. pendula trees growing in the free air humidity manipulation (FAHM) experimental site, where treatments included elevated relative air humidity and elevated soil moisture. A high-performance liquid chromatography-quadrupole-time of flight mass spectrometer (HPLC-qTOF-MS) was used to analyse secondary metabolites. Our results showed accumulation of LSM depends on the canopy position and competitive status. Flavonoids (FLA), dihydroxybenzoic acids (HBA), jasmonates (JA) and terpene glucosides (TG) were higher in the upper canopy, and FLA, monoaryl compounds (MAR) and sesquiterpenoids (ST) were higher in dominant trees. The FAHM treatments had a more distinct effect on RSM than on LSM. The RSMs were lower in elevated air humidity and soil moisture conditions than in control conditions. The RSM content depended on the competitive status and was higher in suppressed trees. Our study suggests that young B. pendula will allocate similar amounts of carbon to constitutive chemical leaf defence, but a lower amount to root defence (per fine root biomass) under higher humidity.

The ant’s weapon improves honey bee learning performance

Formic acid is the main component of the ant’s major weapon against enemies. Being mainly used as a chemical defense, the acid is also exploited for recruitment and trail marking. The repelling effect of the organic acid is used by some mammals and birds which rub themselves in the acid to eliminate ectoparasites. Beekeepers across the world rely on this effect to control the parasitic mite Varroa destructor. Varroa mites are considered the most destructive pest of honey bees worldwide and can lead to the loss of entire colonies. Formic acid is highly effective against Varroa mites but can also kill the honeybee queen and worker brood. Whether formic acid can also affect the behavior of honey bees is unknown. We here study the effect of formic acid on sucrose responsiveness and cognition of honey bees treated at different live stages in field-relevant doses. Both behaviors are essential for survival of the honey bee colony. Rather unexpectedly, formic acid clearly improved the learning performance of the bees in appetitive olfactory conditioning, while not affecting sucrose responsiveness. This exciting side effect of formic acid certainly deserves further detailed investigations.

Tissue-specific plant toxins and adaptation in a specialist root herbivore

In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.