Repeated horizontal acquisition of lagriamide-producing symbionts in Lagriinae beetles.

Microbial symbionts associate with multicellular organisms on a continuum from facultative associations to mutual codependency. In the oldest intracellular symbioses there is exclusive vertical symbiont transmission, and co-diversification of symbiotic partners over millions of years. Such symbionts often undergo genome reduction due to low effective population sizes, frequent population bottlenecks, and reduced purifying selection. Here, we describe multiple independent acquisition events of closely related defensive symbionts followed by genome erosion in a group of Lagriinae beetles. Previous work in Lagria villosa revealed the dominant genome-eroded symbiont of the genus Burkholderia produces the antifungal compound lagriamide, protecting the beetle's eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from seven different host species within Lagriinae from five countries, to unravel the evolutionary history of this symbiotic relationship. In each host, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster. However, we did not find evidence for host-symbiont co-diversification, or for monophyly of the lagriamide-producing symbionts. Instead, our analyses support a single ancestral acquisition of the gene cluster followed by at least four independent symbiont acquisitions and subsequent genome erosion in each lineage. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide gene cluster. Our results, therefore, reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by a high degree of specificity, and highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis.

Scots Pines With Tolerance to Melampsora pinitorqua and Diplodia sapinea Show Distinct Metabolic Profiles.

Diplodia sapinea causes Diplodia tip blight (DTB) and is recognised as an opportunistic necrotrophic pathogen affecting conifers. While DTB is associated with abiotic stress, the impact of biotic stress in the host on D. sapinea's lifestyle shift is unknown. Observed co-occurrences of D. sapinea and Melampsora pinitorqua, causing pine twisting rust on Scots pine (Pinus sylvestris), instigated an investigation into their interaction with and influence on the defence mechanisms of the host. We hypothesised that M. pinitorqua infections predispose the trees to D. sapinea by stressing the host and altering the shoot metabolites. Pines in a plantation were sampled over time to study pathogen biomass and host metabolites. Symptoms of both pathogens were consistent over years, and the preceding season's symptoms affected the metabolic profiles pre-infection and M. pinitorqua's proliferation. Symptoms of M. pinitorqua altered shoot metabolites more than fungal biomass, with co-symptomatic trees exhibiting elevated M. pinitorqua biomass. Specific phenolic compounds had a strong positive association with the shoot symptom × D. sapinea interaction. D. sapinea's biomass presymptoms was independent of previous disease symptoms and infection by M. pinitorqua. Some trees showed disease tolerance, with delayed rust infections and minimal DTB symptoms. Further investigations on this trait are needed.

Intraspecific Variation in the Alkaloids of Adalia decempunctata (Coleoptera, Coccinellidae): Sex, Reproduction and Colour Pattern Polymorphism.

In this paper, we examine intraspecific variation in the quantity of alkaloid chemical defence in field collected individuals of the polymorphic ladybird beetle Adalia decempunctata (10-spot ladybird). Like its more widely studied relative Adalia bipunctata (2-spot ladybird), A. decempunctata possesses the alkaloids adaline and adalinine, which are, respectively, the major and minor alkaloids of A. bipunctata. We focused especially on alkaloid concentration in relation to colour pattern morph, sex, and the relationship between female and egg parameters. There was a marked sexual dimorphism in the balance of the two alkaloids, with adaline predominating in females and adalinine predominating in males: in males, on average, over 70% of total alkaloid was adalinine. Females had a lower proportion of adalinine (< 10%) than their eggs (> 15%) and relationships between egg alkaloid and female alkaloid or fecundity were weak or non-existent. Colour pattern morph had a borderline (although not) significant relationship with adaline concentration and total alkaloid concentration, which could be further explored with laboratory reared individuals. The sexual dimorphism in alkaloid content, which seems likely due to differences in synthesis, might be related to their relative costs to the two sexes and might provide insight into the evolution of alkaloid diversity in ladybirds.

Repeated horizontal acquisition of lagriamide-producing symbionts in Lagriinae beetles.

Microbial symbionts associate with multicellular organisms on a continuum from facultative associations to mutual codependency. In some of the oldest intracellular symbioses there is exclusive vertical symbiont transmission, and co-diversification of symbiotic partners over millions of years. Such symbionts often undergo genome reduction due to low effective population sizes, frequent population bottlenecks, and reduced purifying selection. Here, we describe multiple independent acquisition events of closely related defensive symbionts followed by genome erosion in a group of Lagriinae beetles. Previous work in Lagria villosa revealed the dominant genome-eroded symbiont of the genus Burkholderia produces the antifungal compound lagriamide and protects the beetle's eggs and larvae from antagonistic fungi. Here, we use metagenomics to assemble 11 additional genomes of lagriamide-producing symbionts from seven different host species within Lagriinae from five countries, to unravel the evolutionary history of this symbiotic relationship. In each host species, we detected one dominant genome-eroded Burkholderia symbiont encoding the lagriamide biosynthetic gene cluster (BGC). Surprisingly, however, we did not find evidence for host-symbiont co-diversification, or for a monophyly of the lagriamide-producing symbionts. Instead, our analyses support at least four independent acquisition events of lagriamide-encoding symbionts and subsequent genome erosion in each of these lineages. By contrast, a clade of plant-associated relatives retained large genomes but secondarily lost the lagriamide BGC. In conclusion, our results reveal a dynamic evolutionary history with multiple independent symbiont acquisitions characterized by high degree of specificity. They highlight the importance of the specialized metabolite lagriamide for the establishment and maintenance of this defensive symbiosis.

Chemical defence of a centipede (Clinopodes flavidus).

Chemical substances are of utmost importance for the biotic interactions between animals and their predators/parasites; many of these semiochemicals are emitted for defence purposes. One of the most deterrent and toxic biogenic substances we know of is hydrogen cyanide, which can be stored by certain insects, millipedes, centipedes and arachnids in the form of stable and less volatile molecules. The aim of this study was to analyse the biology and chemistry of such a defence mechanism in a geophilomorph centipede (Chilopoda). The cyanogenic secretion of Clinopodes flavidus is discharged from the ventral glands, whose glandular units are located in the space between the cuticle and the trunk muscles and do not extend deep into the segment. In addition to hydrogen cyanide, the ventral secretion contains 2-methylpentanoic acid, benzaldehyde, benzoyl cyanide, 2-methyl branched C-9 carboxylic acid (tentatively identified as 2-methyloctanoic acid), methyl 2-phenylacetate, benzoic acid and mandelonitrile as well as four major proteins with a molecular weight of 150, 66.2, 59 and 55 kDa. The correlation between the presence of ventral glands and guarding with the female's ventral side facing away from the eggs and young indicates a functional link between these two traits. We hope that the specificity of the chemical composition of the ventral secretion could serve as a criterion for chemotaxonomy and that the analysis of more species will help to clarify the phylogenetic relationships within the Geophilomorpha.

Drug Discovery Potential of Insect-Derived Compounds: A review.

Insects are the most numerous and diverse collection of organisms on earth. Around the world, human societies have utilised insects and the materials derived from them as medical resources. These arthropods use chemistry, like all other species. According to their survival needs, they make adaptations, including protecting themselves from infection as well as predation, being able to connect with a social life, and carrying out generations with survivability in the environment. The main focus of the study of chemical ecology is the abundance of chemical compounds found in arthropods that are used for these ecological adaptations. This thorough analysis summarises the huge potential for finding new natural compounds with medical benefits from the Arthropods. Insects and their derivatives have a wide range of uses, and their "raw products" have made significant strides in a wide range of industries, including pharmaceuticals, tissue engineering, dentistry, plant and agricultural science, veterinary medicine, cosmetics, and cosmeceuticals, food, and nutraceuticals, among others. Bioactive components derived from insects are emerging as potential therapeutic sources that are beneficial against a variety of disorders. Insects, which have a huge variety of species, are an intriguing and potential source of low molecular biologically active natural compounds that are either produced by the insect itself or by accompanying microbes. The present review work collated the updates of insect-derived compounds, the use of insects worldwide, and drug discovery potential of insect derivatives.

High toxin concentration in pollen may deter collection by bees in butterfly-pollinated Rhododendron molle.

BACKGROUNDS AND AIMS: The hypothesis that plants evolve features that protect accessible pollen from consumption by flower visitors remains poorly understood. METHODS: To explore potential chemical defence against pollen consumption, we examined the pollinator assemblage, foraging behaviour, visitation frequency and pollen transfer efficiency in Rhododendron molle, a highly toxic shrub containing rhodojaponin III. Nutrient (protein and lipid) and toxic components in pollen and other tissues were measured. KEY RESULTS: Overall in the five populations studied, floral visits by butterflies and bumblebees were relatively more frequent than visits by honeybees. All foraged for nectar but not pollen. Butterflies did not differ from bumblebees in the amount of pollen removed per visit, but deposited more pollen per visit. Pollination experiments indicated that R. molle was self-compatible, but both fruit and seed production were pollen-limited. Our analysis indicated that the pollen was not protein-poor and had a higher concentration of the toxic compound rhodojaponin III than petals and leaves, this compound was undetectable in nectar. CONCLUSION: Pollen toxicity in Rhododendron flowers may discourage pollen robbers (bees) from taking the freely accessible pollen grains, while the toxin-free nectar rewards effective pollinators, promoting pollen transfer. This preliminary study supports the hypothesis that chemical defence in pollen would be likely to evolve in species without physical protection from pollinivores.

Exotic predators can sequester and use novel toxins from exotic non-coevolved prey.

Defensive chemicals of prey can be sequestered by some coevolved predators, which take advantage of prey toxins for their own defence. The increase in the number of invasive species in the Anthropocene has resulted in new interactions among non-coevolved predator and prey species. While novelty in chemical defence may provide a benefit for invasive prey against non-coevolved predators, resident predators with the right evolutionary pre-adaptations might benefit from sequestering these novel defences. Here, we chose a well-known system of invasive species to test whether non-coevolved predators can sequester and use toxins from exotic prey. Together with the invasive prickly pear plants, cochineal bugs (Dactylopius spp.) are spreading worldwide from their native range in the Americas. These insects produce carminic acid, a defensive anthraquinone that some specialized predators sequester for their own defence. Using this system, we first determined whether coccinellids that prey on cochineal bugs in the Mediterranean region tolerated, sequestered, and released carminic acid in reflex bleeding. Then, we quantified the deterrent effect of carminic acid against antagonistic ants. Our results demonstrate that the Australian coccinellid Cryptolaemus montrouzieri sequestered carminic acid, a substance absent in its coevolved prey, from exotic cochineal bugs. When attacked, the predator released this substance through reflex bleeding at concentrations that were deterrent against antagonistic ants. These findings reveal that non-coevolved predators can sequester and use novel toxins from exotic prey and highlights the surprising outcomes of novel interactions that arise from species invasions.

Phylosymbiosis and metabolomics resolve phenotypically plastic and cryptic sponge species in the genus Agelas across the Caribbean basin.

Fundamental to holobiont biology is recognising how variation in microbial composition and function relates to host phenotypic variation. Sponges often exhibit considerable phenotypic plasticity and also harbour dense microbial communities that function to protect and nourish hosts. One of the most prominent sponge genera on Caribbean coral reefs is Agelas. Using a comprehensive set of morphological (growth form, spicule), chemical and molecular data on 13 recognised species of Agelas in the Caribbean basin, we were able to define only five species (=clades) and found that many morphospecies designations were incongruent with phylogenomic and population genetic analyses. Microbial communities were also strongly differentiated between phylogenetic species, showing little evidence of cryptic divergence and relatively low correlation with morphospecies assignment. Metagenomic analyses also showed strong correspondence to phylogenetic species, and to a lesser extent, geographical and morphological characters. Surprisingly, the variation in secondary metabolites produced by sponge holobionts was explained by geography and morphospecies assignment, in addition to phylogenetic species, and covaried significantly with a subset of microbial symbionts. Spicule characteristics were highly plastic, under greater impact from geographical location than phylogeny. Our results suggest that while phenotypic plasticity is rampant in Agelas, morphological differences within phylogenetic species affect functionally important ecological traits, including the composition of the symbiotic microbial communities and metabolomic profiles.

Influences of compound age and identity in the effectiveness of insect quinone secretions against the fungus Beauveria bassiana.

Chemical defences against parasites and pathogens can be seen in a wide range of animal taxa, including insect pests such as the red flour beetle Tribolium castaneum. Antimicrobial quinone-based secretions can be used by these beetles to defend against various parasites, particularly the fungal entomopathogen Beauveria bassiana. While quinone secretions can inhibit B. bassiana growth, it is unknown how long they remain effective or how individual secretion compounds contribute to growth inhibition. Here, we tested each individual component of the quinone secretions (methyl-1,4-benzoquinone, ethyl-1,4-benzoquinone, and 1-pentadecene), as well as two mixed solutions that represent the composition range found in natural T. castaneum secretions, after aging for 0, 24, or 72 h. The two quinone compounds equally contributed to B. bassiana inhibition, but their efficacy was significantly reduced after 24 h, with no growth inhibition after 72 h. This indicates that quinones protect insects against B. bassiana for only a limited time, perhaps requiring constant secretion into the environment to effectively defend against this fungal threat. Future investigations may consider the extent to which quinone secretions are effective against other parasites, as well as how their ability to cause parasite damage changes with compound age.

Soil cadmium pollution facilitated the invasion of alligator weed through enhanced herbivore resistance and competitive ability over a congeneric species.

A number of invasive plant species, such as Alternanthera philoxeroides, have been documented to be able to accumulate trace metal elements in their tissues. Since metal accumulation in plants can serve as a defence against herbivores, we hypothesized that metal pollution will increase herbivore resistance of metal-accumulating invasive plant species and such a benefit will grant them a competitive advantage over local co-occurring plants. In this study, we compared the differences in plant growth and herbivore feeding preference between A. philoxeroides and its native congener Alternanthera sessilis in single and mixed cultures with and without soil cadmium (Cd) pollution. The results showed that A. philoxeroides plants were more tolerant to Cd stress and accumulated more Cd in the leaves than A. sessilis. Cd exposure increased the resistance of A. philoxeroides against a specialist and a generalist herbivore compared with A. sessilis. Competition experiments indicated that Cd stress largely increased the competitive advantage of A. philoxeroides over A. sessilis with or without herbivore pressures. The differences in herbivore resistance between the two plant species under soil Cd stress are most likely due to the deterring effect of Cd accumulation and Cd-enhanced mechanical defences rather than changes in leaf specialized metabolites.

Aristolochia mimics stink bugs to repel vertebrate herbivores via TRPA1 activation.

Mimicry is the phenomenon in which one species (the mimic) closely resembles another (the model), enhancing its own fitness by deceiving a third party into interacting with it as if it were the model. In plants, mimicry is used primarily to gain fitness by withholding rewards from mutualists or deterring herbivores cost-effectively. While extensive work has been documented on putative defence mimicry, limited investigation has been conducted in the field of chemical mimicry. In this study, we used field experiments, chemical analyses, behavioural assays, and electrophysiology, to test the hypothesis that the birthwort Aristolochia delavayi employs chemical mimicry by releasing leaf scent that closely resembles stink bug defensive compounds and repels vertebrate herbivores. We show that A. delavayi leaf scent is chemically and functionally similar to the generalized defensive volatiles of stink bugs and that the scent effectively deters vertebrate herbivores, likely through the activation of TRPA1 channels via (E)-2-alkenal compounds. This study provides an unequivocal example of chemical mimicry in plants, revealing intricate dynamics between plants and vertebrate herbivores. Our study underscores the potency of chemical volatiles in countering vertebrate herbivory, urging further research to uncover their potentially underestimated importance.

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.

No-cost meals might not exist for insects feeding on toxic plants.

Plants produce chemicals (or plant specialised/secondary metabolites, PSM) to protect themselves against various biological antagonists. Herbivorous insects use plants in two ways: as a food source and as a defence source. Insects can detoxify and sequester PSMs in their bodies as a defence mechanism against predators and pathogens. Here, I review the literature on the cost of PSM detoxification and sequestration in insects. I argue that no-cost meals might not exist for insects feeding on toxic plants and suggest that potential costs could be detected in an ecophysiological framework.

Experimental Growth Conditions affect Direct and Indirect Defences in two Cotton Species.

Cotton has been used as a model plant to study direct and indirect plant defence against herbivorous insects. However, the plant growing conditions could have an important effect on the outcome of such plant defence studies. We examined how common experimental growth conditions influence constitutive and inducible defences in two species of cotton, Gossypium hirsutum and G. herbaceum. We induced plants by applying caterpillar regurgitant to mechanical wounds to compare the induction levels between plants of both species grown in greenhouse or phytotron conditions. For this we measured defence metabolites (gossypol and heliocides) and performance of Spodoptera frugiperda caterpillars on different leaves, the emission of plant volatiles, and their attractiveness to parasitic wasps. Induction increased the levels of defence metabolites, which in turn decreased the performance of S. frugiperda larvae. Constitutive and induced defence levels were the highest in plants grown in the phytotron (compared to greenhouse plants), G. hirsutum and young leaves. Defence induction was more pronounced in plants grown in the phytotron and in young leaves. Also, the differences between growing conditions were more evident for metabolites in the youngest leaves, indicating an interaction with plant ontogeny. The composition of emitted volatiles was different between plants from the two growth conditions, with greenhouse-grown plants showing more variation than phytotron-grown plants. Also, G. hirsutum released higher amounts of volatiles and attracted more parasitic wasps than G. herbaceum. Overall, these results highlight the importance of experimental abiotic factors in plant defence induction and ontogeny of defences. We therefore suggest careful consideration in selecting the appropriate experimental growing conditions for studies on plant defences.

Rapid but narrow - Evolutionary adaptation and transcriptional response of Drosophila melanogaster to toxic mould.

Insects have adapted to a multitude of environmental conditions, including the presence of xenobiotic noxious substances. Environmental microorganisms, particularly rich on ephemeral resources, employ these noxious chemicals in a chemical warfare against predators and competitors, driving co-evolutionary adaptations. In order to analyse how environmental microbes may be driving such evolutionary adaptations, we experimentally evolved Drosophila melanogaster populations by exposing larvae to the toxin-producing mould Aspergillus nidulans that infests the flies' breeding substrate. To disentangle the effects of the mycotoxin Sterigmatocystin from other substrate modifications inflicted by the mould, we used the following four selection regimes: (i) control without fungus, (ii) A. nidulans wild type, (iii) a mutant of A. nidulans ΔlaeA with impaired toxin production, (iv) synthetic Sterigmatocystin. Experimental evolution was carried out in five independent D. melanogaster populations each, for a total of 11 generations. We further combined our evolution experiment with transcriptome analysis to identify evolutionary shifts in gene expression due to the selection regimes and mould confrontation. Populations that evolved in presence of the toxin-producing mould or the pure mycotoxin rapidly adapted to the respective conditions and showed higher viability in subsequent confrontations. Yet, mycotoxin-selected populations had no advantage in A. nidulans wild type confrontation. Moreover, distinctive changes in gene expression related to the selection-regime contrast were only associated with the toxin-producing-fungus regime and comprised a narrow set of genes. Thus, it needs the specific conditions of the selection agent to enable adaptation to the fungus.

Evolutionarily stable levels of aposematic defence in prey populations.

Our understanding of aposematism (the conspicuous signalling of a defence for the deterrence of predators) has advanced notably since its first observation in the late nineteenth century. Indeed, it extends the scope of a well-established game-theoretical model of this very same process both from the analytical standpoint (by considering regimes of varying background mortality and colony size) and from the practical standpoint (by assessing its efficacy and limitations in predicting the evolution of prey traits in finite simulated populations). The nature of the manuscript at hand is more mathematical and its aim is two-fold: first, to determine the relationship between evolutionarily stable levels of defence and signal strength under various regimes of background mortality and colony size. Second, to compare these predictions with simulations of finite prey populations that are subject to random local mutation. We compare the roles of absolute resident fitness, mutant fitness and stochasticity in the evolution of prey traits and discuss the importance of population size in the above.

Extreme acidity in a cynipid gall: a potential new defensive strategy against natural enemies.

The morphology of insect-induced galls contributes to defences of the gall-inducing insect species against its natural enemies. In terms of gall chemistry, the only defensive compounds thus far identified in galls are tannins that accumulate in many galls, preventing damage by herbivores. Intrigued by the fruit-like appearance of the translucent oak gall (TOG; Amphibolips nubilipennis, Cynipidae, Hymenoptera) induced on red oak (Quercus rubra), we hypothesized that its chemical composition may deviate from other galls. We found that the pH of the gall is between 2 and 3, making it among the lowest pH levels found in plant tissues. We examined the organic acid content of TOG and compared it to fruits and other galls using high-performance liquid chromatography and gas chromatography-mass spectrometry. Malic acid, an acid with particularly high abundance in apples, represents 66% of the organic acid detected in TOGs. The concentration of malic acid was two times higher than in other galls and in apples. Gall histology showed that the acid-containing cells were enlarged and vacuolized just like fruits mesocarp cells. Accumulation of organic acid in gall tissues is convergent with fruit morphology and may constitute a new defensive strategy against predators and parasitoids.

Intrinsic Factors Associated with Dietary Toxin Quantity and Concentration in the Nuchal Glands of a Natricine Snake Rhabdophis Tigrinus.

The snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads and stores them in the nuchal glands as defensive toxins. It has previously been shown that there are individual differences in the total quantity of BDs stored in the nuchal glands of adult R. tigrinus and that BD quantities and profiles of R. tigrinus exhibit geographic variation. However, no previous study has examined the total quantity of BDs as a percentage of body mass (relative BD quantity) and the concentration of BDs in the nuchal gland fluid (BD gland concentration). In addition, intrinsic factors that are associated with relative BD quantity and BD concentration have not been examined within a single population. We collected 158 adult snakes from an area of central Japan from May to October and analyzed their BD quantities by UV analysis. We assessed individual differences in BD quantity, relative BD quantity and BD gland concentration. We found that 1) in approximately 60% of the 158 individuals, the BD gland concentration was greater than 50%; 2) body length and body condition are positively correlated with relative BD quantity and BD gland concentration; 3) even in a single population, individual differences of BD quantity are large, and are greater in females than in males; and 4) relative BD quantity and BD gland concentration of females during the gestation season are lower than those during the non-gestation season.