Chemical Defense Mechanisms Research Articles

Extracting and Analyzing Pyrrolizidine Alkaloids in Medicinal Plants: A Review.

Pyrrolizidine alkaloids (PAs) are distributed in plant families of Asteraceae, Boraginaceae, and Fabaceae and serve in the chemical defense mechanism against herbivores. However, they became a matter of concern due to their toxicity associated with the high risk of intake within herbal preparations, e.g., phytopharmaceutical formulations, medicinal teas, or other plant-derived drug products. In 1992, the German Federal Ministry of Health established the first limits of PA content for fourteen medicinal plants. Because of the toxic effects of PAs, the Federal Institute of Risk Assessment (BfR) established more stringent limits in 2011, whereby a daily intake <0.007 µg/kg body weight was recommended and valid until 2018. A threefold higher limit was then advised by BfR. To address consumer safety, there is the need for more efficient extraction procedures along with robust, selective, and sensitive analytical methods to address these concerns. With the increased prevalence of, e.g., phytopharmaceutical formulations, this timely review comprehensively focuses on the most relevant extraction and analysis strategies for each of those fourteen plant genera. While a variety of extraction procedures has been reported, differences in PA content of up to 1110 ppm (0.11% (w/w)) were obtained dependent on the nature of the solvent and the applied extraction technique. It is evident that the efficient extraction of PAs requires further improvements or at least standardization of the extraction conditions. Comparing the various analytical techniques applied regarding selectivity and sensitivity, LC-MS methods appear most suited. This review shows that both standardized extraction and sensitive determination of PAs is required for achieving appropriate safety levels concerning public health in future.

Hydrophilic hierarchical carbon with TiO2 nanofiber membrane for high separation efficiency of dye and oil-water emulsion

Hierarchical carbon hybrid materials have gained more attention in recent times especially in the separation of pollutants from wastewater. In this study, a novel ternary composite of graphitic carbon nitride (G-C3N4) decorated on reduced graphene oxide (RGO) with titanium dioxide (TiO2) nanohybrids was synthesized using hydrothermal approach to modify the electrospun PVDF membranes for the separation of oil and dye in wastewater. The characteristic functional groups of G-C3N4, RGO and TiO2 (G-C3N4,/RGO/TiO2) nanohybrids were confirmed by X-ray diffraction (XRD) and X-ray photon electron spectroscopy. Moreover, the prepared membranes were also studied using field emission scanning electron microscope and X-ray photoelectron spectroscopy. Interestingly, the PVDF modified membranes displayed excellent thermal stability with lower contact angle, increased surface roughness and surface charges. It was due to the tunable covalent π-π interfaces among G-C3N4 and RGO 2D hierarchical structured nanosheets. Besides due to the reasonable hierarchical structure formation of chemical and physical defense mechanism, the modified membranes exhibited outstanding antifouling performance and different oil water emulsion rejections. The pure water flux for GT-4 membrane was around 1261.2 ± 2 L/m2.h and exhibited high oil water removal efficiency above 95.4 ± 0.1% for different oils. The modified PVDF membranes also exhibited higher rejection rate of about 94.2 ± 0.5% for methylene blue dye removals. These studies confirm the formation of hydration layer and hierarchical structure for membranes which can be employed in oil water emulsion separation and dye rejection studies.

Preservation of monoterpenoids in Oligocene resin: Insights into the evolution of chemical defense mechanism of plants in deep-time

Monoterpenoid class of compounds is well-known for the characteristic odor and their contribution in various ecological functions such as plant-insect interaction, defense, pollinator attraction etc. They are extremely volatile and labile by nature and therefore their preservation in geological samples is quite uncommon. In the present article we report, exceptionally preserved monoterpenoids in late Oligocene resin from offshore Shimokita, Japan. Compounds viz. fenchol, fenchone, camphor, borneol, isoborneol are detected in appreciable quantity while camphene, isocamphene and p-cymene are found in low amount. Such rare phenomenon suggests that the amber must have experienced some unique diagenetic condition which favored the preservation of these volatile compounds. These monoterpenes have distinct aroma and they actively contribute into the chemical defense mechanisms of extant plant species. Their excellent preservation in late Oligocene amber depicts that such chemical defense strategies had already been developed by plants during early Cenozoic.

Epiphytic diatom assemblages on invasive Caulerpa taxifolia and autochthonous Halimeda tuna and Padina sp. seaweeds in the Adriatic Sea – summer/autumn aspect

Abstract This study focuses on the taxonomy of epiphytic diatoms in the area of invasive macroalgae from the genus Caulerpa. Caulerpa species are characterized by the presence of secondary metabolites, such as caulerpenyne (CYN), the main function of which is a chemical defense mechanism against herbivores and epiphytes. Epiphytic diatoms were studied on fronds of Caulerpa taxifolia (“killer seaweed”) and, for comparison, on autochthonous macroalgae Padina sp. and Halimeda tuna at the eastern Adriatic Sea coast (Island of Hvar) in the summer and autumn of 2010. The qualitative analysis was performed with the use of light and scanning electron microscopy. The Shannon–Wiener Diversity Index determined for Caulerpa taxifolia showed a wide range of values (3.11–4.88), with a maximum in August and a minimum in October. While the number of taxa on Caulerpa taxifolia fronds increased from June (41) to August (88), it declined in autumn due to the high relative abundance of Cocconeis caulerpacola, which is a diatom typical for Caulerpa. On the other hand, the largest number of taxa on Padina sp. was observed in September (82). The detailed composition of epiphytic diatoms and seasonal dynamics in the area affected by the invasive macroalga Caulerpa taxifolia have been determined for the first time.

Biogenic volatiles of rupicolous plants act as direct defenses against molluscs: The case of the endangered Clinopodium rouyanum

Clinopodium rouyanum (Briq.) Govaerts is an endemic species from the mountains of the island of Majorca. In order to assess chemical defense mechanisms of this plant against molluscs, a chemical composition analysis of the essential plant oil (obtained by distillation SDE) and volatiles emitted by green parts, flowers, corollas and tube corollas (obtained by SPME and recent ST methods) – analyzed by GC and GC-MS – was conducted. Additionally, a study on the morphological types and distribution of trichomes was carried out by electron scanning microscopy. Also, the plant’s deterrence to snails was evaluated. The essential oil and volatiles were characterized by a high concentration of oxygenated monoterpenes (83.3–99.9%), whose principal compounds were Pulegone (73.0–93.9%) and Menthone (3.1%–11.8%). Non-glandular tricellular trichomes, peltate trichomes, and capitate trichomes were observed on leaves, calyx, corolla and stems. Volatile components of plants are effective deterrents to a generalist molluscan herbivore and represent an effective defense communication system that favors the conservation of this endangered species.

Surface chemical defence of the eelgrass Zostera marina against microbial foulers

Plants rely on both mechanical and chemical defence mechanisms to protect their surfaces against microorganisms. The recently completed genome of the eelgrass Zostera marina, a marine angiosperm with fundamental importance for coastal ecosystems, showed that its re-adaptation from land to the sea has led to the loss of essential genes (for chemical communication and defence) and structural features (stomata and thick cuticle) that are typical of terrestrial plants. This study was designed to understand the molecular nature of surface protection and fouling-control strategy of eelgrass against marine epiphytic yeasts. Different surface extraction methods and comparative metabolomics by tandem mass spectrometry (LC-MS/MS) were used for targeted and untargeted identification of the metabolite profiles of the leaf surface and the whole tissue extracts. Desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) coupled with traditional bioassays revealed, for the first time, the unique spatial distribution of the eelgrass surface-associated phenolics and fatty acids, as well as their differential bioactivity against the growth and settlement of epiphytic yeasts. This study provides insights into the complex chemical defence system of the eelgrass leaf surface. It suggests that surface-associated metabolites modulate biotic interactions and provide chemical defence and structural protection to eelgrass in its marine environment.

Understanding the Cross-Talk of Redox Metabolism and Fe-S Cluster Biogenesis in Leishmania Through Systems Biology Approach.

Leishmania parasites possess an exceptional oxidant and chemical defense mechanism, involving a very unique small molecular weight thiol, trypanothione (T[SH]2), that helps the parasite to manage its survival inside the host macrophage. The reduced state of T[SH]2 is maintained by NADPH-dependent trypanothione reductase (TryR) by recycling trypanothione disulfide (TS2). Along with its most important role as central reductant, T[SH]2 have also been assumed to regulate the activation of iron-sulfur cluster proteins (Fe/S). Fe/S clusters are versatile cofactors of various proteins and execute a much broader range of essential biological processes viz., TCA cycle, redox homeostasis, etc. Although, several Fe/S cluster proteins and their roles have been identified in Leishmania, some of the components of how T[SH]2 is involved in the regulation of Fe/S proteins remains to be explored. In pursuit of this aim, a systems biology approach was undertaken to get an insight into the overall picture to unravel how T[SH]2 synthesis and reduction is linked with the regulation of Fe/S cluster proteins and controls the redox homeostasis at a larger scale. In the current study, we constructed an in silico kinetic model of T[SH]2 metabolism. T[SH]2 reduction reaction was introduced with a perturbation in the form of its inhibition to predict the overall behavior of the model. The main control of reaction fluxes were exerted by TryR reaction rate that affected almost all the important reactions in the model. It was observed that the model was more sensitive to the perturbation introduced in TryR reaction, 5 to 6-fold. Furthermore, due to inhibition, the T[SH]2 synthesis rate was observed to be gradually decreased by 8 to 14-fold. This has also caused an elevated level of free radicals which apparently affected the activation of Fe/S cluster proteins. The present kinetic model has demonstrated the importance of T[SH]2 in leishmanial cellular redox metabolism. Hence, we suggest that, by designing highly potent and specific inhibitors of TryR enzyme, inhibition of T[SH]2 reduction and overall inhibition of most of the downstream pathways including Fe/S protein activation reactions, can be accomplished.

The Precursor Hypothesis of Sponge Kleptocnidism: Development of Nematocysts in Haliclona cnidata sp. nov. (Porifera, Demospongiae, Haplosclerida)

Marine sponges thrive in benthic environments despite intense spatial competition and predator pressure. The sessile filter feeders usually compensate their lack of physical defense and behavioral escape by a high level of bioactivity. In the stinging black sponge (Haliclona cnidata sp. nov.), these chemical defense mechanisms are complemented by ‘cellular weapons’ – functional nematocysts likely acquired from cnidarians (kleptocnidism). Whereas kleptocnidism might be facilitated by a close contact with cnidarian donors, preliminary investigations suggest that the stinging black sponge sustains nematocysts even if kept apart from cnidarians. As the underlying mechanisms causing this phenomenon remain unknown, the development of its nematocysts was studied in both presence and absence of potential cnidarian donors. First, we compared inherent nematocysts of adult sponge individuals with foreign nematocysts of co-cultivated cnidarians to identify potential donors. Second, we experimentally assessed the donor independent and donor dependent development of inherent and foreign nematocysts within cultures of sponge cell aggregates (SCAs). The inherent nematocysts comprised two specific types that both differed from those of the eight co-cultivated cnidarians. Specifically, we showed that the number of sponge-inherent nematocysts increased in SCAs over time in the absence of potential donors. Numbers of supplied foreign nematocysts, however, did not increase in the SCAs. We conclude that the observed increase of inherent kleptocnidae nematocysts is due to the maturation of nematocyst precursor cells. Given these findings, we here propose the precursor hypothesis of sponge kleptocnidism. Accordingly, nematocyst precursors or immature nematocyte-nematocyst complexes might be initially acquired by sponges through filtration, maintained in sponge tissues, and nurtured to fully functioning nematocyte-nematocyst complexes. The underlying evolutionary processes are likely facilitated by bacteria derived secondary metabolites and photosynthetically active dinoflagellates. Due to a simple body plan and the in vitro proliferation capacity of sponge cells, Haliclona cnidata sp. nov. may serve as a novel evolutionary model system to further assess fundamental research questions regarding kleptocnidism. This study not only sheds new light on kleptocnidism in sponges, it also calls for a holobiontic view at defense mechanisms that involves the actual sponge, cnidarian nematocysts, dinoflagellate endosymbionts, and a complex microbial community.

High niche diversity in Mesozoic pollinating lacewings

Niche diversity of pollinating insects plays a vital role in maintaining extant terrestrial ecosystems. A key dimension of pollination niches refers to the insect proboscis length that commonly matches the floral tube length. Here we describe new kalligrammatid lacewings (an iconic Mesozoic pollinating insect lineage) from late Cretaceous Burmese amber and Mesozoic sediments in China. Kalligrammatids display complex configurations of elongate mouthpart elements consisting of well-developed maxillae, labium and their palps. The mouthpart lengths vary among species, from 0.6 to 18.0 mm, suggesting corresponding variability in the floral tube lengths of Mesozoic plants. With the diversification of pollinating habits, the kalligrammatids presented highly divergent traits related to chemical communication and defence mechanisms. Together with other Mesozoic long-proboscid insects, these fossils not only reveal the high niche diversity of Mesozoic pollinating insects but also highlight the diversity of Mesozoic pollinator-dependent plants prior to the rise of angiosperms.

Application of Direct Analysis in Real Time–High Resolution Mass Spectrometry to Investigations of Induced Plant Chemical Defense Mechanisms—Revelation of Negative Feedback Inhibition of an Alliinase

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 μM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.

How different sterols contribute to saponin tolerant plasma membranes in sea cucumbers

Sea cucumbers produce saponins as a chemical defense mechanism, however their cells can tolerate the cytotoxic nature of these chemicals. To elucidate the molecular mechanisms behind this tolerance a suite of complementary biophysical tools was used, firstly using liposomes for in vitro techniques then using in silico approaches for a molecular-level insight. The holothuroid saponin Frondoside A, caused significantly less permeabilization in liposomes containing a Δ7 holothuroid sterol than those containing cholesterol and resulted in endothermic interactions versus exothermic interactions with cholesterol containing liposomes. Lipid phases simulations revealed that Frondoside A has an agglomerating effect on cholesterol domains, however, induced small irregular Δ7 sterol clusters. Our results suggest that the structural peculiarities of holothuroid sterols provide sea cucumbers with a mechanism to mitigate the sterol-agglomerating effect of saponins, and therefore to protect their cells from the cytotoxicity of the saponins they produce.

Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen.

Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline “M73,” with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2–3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to wheat. Results are consistent with the hypotheses that (1) dormant seeds are capable of mounting complex responses to pathogens, (2) a diversity of defense enzymes are involved in responses in multiple plant species, and (3) it is possible to identify fungi capable of selective decay of weed seeds without damaging crop seeds, a concept that may be applicable to weed management in the field. While earlier work on seed defenses demonstrated the presence of passive defenses, this work shows that dormant seeds are also quite responsive and capable of activating and releasing defense enzymes in response to a pathogen.

Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels.

Guanidinium toxins, such as saxitoxin (STX), tetrodotoxin (TTX) and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV). Members of the STX group, known collectively as paralytic shellfish toxins (PSTs), are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards development of pharmaceuticals and therapeutic applications.

Impacts of soybean-induced defenses on Spodoptera frugiperda (Lepidoptera: Noctuidae) development

As soybean (Glycine max [L.] Merrill) is an important crop throughout the world, the action of herbivorous insects responsible for economic and productivity losses is the subject of constant research. Spodoptera frugiperda (J.E. Smith) caterpillars can cause extensive damage to soybean culture; this work investigated possible harmful effects on these caterpillars associated with the possible induced defenses of soybean plants. For this purpose, we assessed the biology of the insect (leaf consumption and performance traits) and chemical composition of the soybean leaves by ultraviolet–visible spectroscopy and chemometrics of three treatments: control, mechanically wounded soybean leaves, and S. frugiperda-damaged soybean leaves. The results reveal that both types of injuries induce changes in soybean metabolism regarding the production of phenolic substances, although only the herbivore-damaged plants provoke negative effects on insect biology. Variations in carotenoid production during the circadian cycle were also found in the control group. These results confirm that the soybean plants could endure and activate chemical defense mechanisms that impair the developmental lifecycle of the insect, suggesting possibilities for sustainable control strategies.

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)-How Does the Endoplasmic Reticulum Membrane Influence Their Function?

Uridine diphosphate-glucuronosyltransferases (UGTs) are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER) of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes). They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body’s chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon.

Predator–prey interaction between greenhead ants and processionary caterpillars is mediated by chemical defence

The predatory greenhead ant, Rhytidoponera metallica, readily attacks some insects but not the processionary caterpillar, Ochrogaster lunifer. This urticarial lepidopteran larva is covered in long hairs and has numerous smaller, detachable barbed setae present on the third instar and older larvae. These setae are a health risk to humans and other mammals. In this study, we tested whether O. lunifer larvae are defended against invertebrate predators, using R. metallica as an example. Field experiments at R. metallica nests and laboratory olfactometer experiments were used to assess possible behavioural, morphological and chemical defence mechanisms. We found that a volatile chemical component from O. lunifer larvae inhibited attack/approach by R. metallica. This odour was associated with O. lunifer cuticular components: larval hairs, setae and exoskeleton. Behavioural defence was not found to inhibit R. metallica attack. Furthermore, despite the dense hairs and setae covering O. lunifer larvae, these did not act as a physical barrier to ant attack. Our study demonstrates the key role of chemical ecology in invertebrate interactions involving such hairy caterpillars. Future isolation of the deterrent chemical(s) produced by O. lunifer larvae may offer novel possibilities for managing ant behaviour.

Experimental evidence of chemical defence mechanisms in Antarctic bryozoans

Bryozoans are among the most abundant and diverse members of the Antarctic benthos, however the role of bioactive metabolites in ecological interactions has been scarcely studied. To extend our knowledge about the chemical ecology of Antarctic bryozoans, crude ether extracts (EE) and butanol extracts (BE) obtained from two Antarctic common species (Cornucopina pectogemma and Nematoflustra flagellata), were tested for antibacterial and repellent activities. The extracts were screened for quorum quenching and antibacterial activities against four Antarctic bacterial strains (Bacillus aquimaris, Micrococcus sp., Oceanobacillus sp. and Paracoccus sp.). The Antarctic amphipod Cheirimedon femoratus and the sea star Odontaster validus were selected as sympatric predators to perform anti-predatory and substrate preference assays. No quorum quenching activity was detected in any of the extracts, while all EE exhibited growth inhibition towards at least one bacterium strain. Although the species were not repellent against the sea star, they caused repellence to the amphipods in both extracts, suggesting that defence activities against predation derive from both lipophilic and hydrophilic metabolites. In the substrate preference assays, one EE and one BE deriving from different specimens of the species C. pectogemma were active. This study reveals intraspecific variability of chemical defences and supports the fact that chemically mediated interactions are common in Antarctic bryozoans as means of protection against fouling and predation.

Blepharismins used for chemical defense in two ciliate species of the genus Blepharisma, B. stoltei and B. undulans (Ciliophora: Heterotrichida)

It is known that the freshwater heterotrich ciliate Blepharisma japonicum uses five pigments called blepharismins, stored in its extrusive pigment granules, for both light perception and chemical defense against predators. In this work we focused our attention on the defensive strategies of two additional pigmented species of Blepharisma, B. stoltei and B. undulans. In particular: (1) we observed the predator–prey interactions of B. stoltei or B. undulans against one multicellular and two unicellular predators; (2) we clarified the nature of B. stoltei and B. undulans pigments by means of High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LC-MS); and (3) we demonstrated and compared the toxicity of the purified pigments on a panel of ciliated protists, and against one metazoan predator. The results indicate that the chemical defense mechanism present in B. stoltei and B. undulans is mediated by the same five blepharismins previously characterized for B. japonicum, although produced in different proportions.

Genetic stability of physiological responses to defoliation in a eucalypt and altered chemical defence in regrowth foliage.

Defoliation may initiate physiological recovery and chemical defence mechanisms that allow a plant to improve fitness after damage. Such responses may result in changes in plant resource allocation that influence growth and foliar chemistry. In this study, we investigated the nature and stability of the defoliation response of juvenile plants from three divergent populations of Eucalyptus globulus Labill. A partial defoliation treatment that removed all upper crown leaves and the apical buds was applied to plants sourced from eight families from each of three populations representing contrasting chemical resistance to mammalian herbivory. Growth, photosynthetic rate and chlorophyll content were assessed pre-defoliation and periodically up to 12 weeks post-defoliation. The content of key plant primary and secondary metabolites was assessed pre-defoliation, at 12 weeks post-defoliation in the old foliage (positioned below the point of defoliation) and in the new foliage of the control plants and regrowth (from axillary buds) on the defoliated plants. There were clear treatment impacts on physiological responses, growth and foliar chemical traits, but despite significant constitutive differences in physiology, growth and chemistry the three E. globulus populations did not vary in their response to foliage loss. Distinct physiological responses to defoliation were observed with treatment plants showing significant up-regulation of photosynthetic rate and increased chlorophyll content in the old foliage remaining in the lower crown. There was a significant increase in the concentrations of a number of foliar chemical compounds in the regrowth arising from previously dormant axillary buds compared with new growth derived from apical meristems. There were changes in biomass allocation; defoliated plants had increased branching and leaf biomass, with changes in regrowth morphology to increase light capture. This study argues for multiple responses of E. globulus juveniles to defoliation involving apical bud loss, including elevated chemical defences matched with increased growth. From a chemical defence perspective, these responses create an enhanced chemical mosaic to the herbivore, with leaves remaining after partial browsing potentially being more palatable than the regrowth. This study demonstrates the multiple independent strategies plants may use to respond to partial defoliation and emphasizes the dynamic interplay between growth and defence in the recovery response.

Elucidating a chemical defense mechanism of Antarctic sponges: A computational study

In 2000, a novel secondary metabolite (erebusinone, Ereb) was isolated from the Antarctic sea sponge, Isodictya erinacea. The bioactivity of Ereb was investigated, and it was found to inhibit molting when fed to the arthropod species Orchomene plebs. Xanthurenic acid (XA) is a known endogenous molt regulator present in arthropods. Experimental studies have confirmed that XA inhibits molting by binding to either (or both) of two P450 enzymes (CYP315a1 or CYP314a1) that are responsible for the final two hydroxylations in the production of the molt-inducing hormone, 20-hydroxyecdysone (20E). The lack of crystal structures and biochemical assays for CYP315a1 or CYP314a1, has prevented further experimental exploration of XA and Ereb's molt inhibition mechanisms. Herein, a wide array of computational techniques – homology modeling, molecular dynamics simulations, binding site bioinformatics, flexible receptor–flexible ligand docking, and molecular mechanics-generalized Born surface area calculations – have been employed to elucidate the structure–function relationships between the aforementioned P450s and the two described small molecule inhibitors (Ereb and XA). Results indicate that Ereb likely targets CYP315a1 by interacting with a network of aromatic residues in the binding site, while XA may inhibit both CYP315a1 and CYP314a1 because of its aromatic, as well as charged nature.