GC-MS-based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect.

Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Effects of Host Plants on Development and Immunity of a Generalist Insect Herbivore.

Secondary plant chemistry mediates a variety of communication signals among species, playing a fundamental role in the evolutionary diversification of communities and ecosystems. Herein, we explored diet-mediated host plant effects on development and immune response of a generalist insect herbivore. Vanessa cardui (Nymphalidae) caterpillars were reared on leaves of three host plants that vary in secondary metabolites, Plantago lanceolata (Plantaginaceae), Taraxacum officinale (Asteraceae) and Tithonia diversifolia (Asteraceae). Insect development was evaluated by larval and pupal viabilities, survivorship, and development rate. Immune response was measured as phenoloxidase (PO) activity. Additionally, chemical profiles of the host plants were obtained by liquid chromatograph-mass spectrometry (LC-MS) and the discriminant metabolites were determined using a metabolomic approach. Caterpillars reared on P. lanceolata exhibited the highest larval and pupal viabilities, as well as PO activity, and P. lanceolata leaves were chemically characterized by the presence of iridoid glycosides, phenylpropanoids and flavonoids. Taraxacum officinale leaves were characterized mainly by the presence of phenylpropanoids, flavones O-glycoside and germacranolide-type sesquiterpene lactones; caterpillars reared on this host plant fully developed to the adult stage, however they exhibited lower larval and pupal viabilities compared to individuals reared on P. lanceolata. Conversely, caterpillars reared on T. diversifolia leaves, which contain phenylpropanoids, flavones and diverse furanoheliangolide-type sesquiterpene lactones, were not able to complete larval development and exhibited the lowest PO activity. These findings suggested that V. cardui have adapted to tolerate potentially toxic metabolites occurring in P. lanceolata (iridoid glycosides), however caterpillars were not able to cope with potentially detrimental metabolites occurring in T. diversifolia (furanoheliangolides). Therefore, we suggest that furanoheliangolide-type sesquiterpene lactones were responsible for the poor development and immune response observed for caterpillars reared on T. diversifolia.

Preservation of phenolic compounds on dried leaf infusion of Bauhinia forficata Link

Abstract Bauhinia forficata Link aqueous extract is usually recommended as a phytomedicine to reduce blood glucose levels and its biological activity has been linked to the presence of phenolic compounds from B. forficata preparations. Several drying processes are used in the production of dry herbal extracts, which may influence the chemical composition and efficacy of final herbal medicines. Due to significant chemical changes, defining appropriate drying processes is essential for phytopharmaceutical drug development. In view of this, we analyzed dried B. forficata leaf infusion (BFLI) extracts by HPLC-UV-MSn, followed by molecular networking analysis to evaluate the chemical profiles from dried extracts yielded by freeze-and spray-drying processes. The main metabolites detected included 11 ferulic/isoferulic acid derivatives and 13 glycosylated flavonoids. The qualitative chemical profiles were alike for both drying processes, whereas the relative abundance of some flavonoids was higher using spray-drying. Taken together, our results showed that freeze-and spray-drying preserved the phenolic profile of BFLI and suggested that spray-drying may be the most suitable to obtain its dried products. Along with studying the chemical profiles of dried herbal extracts, evaluating the influence of drying processes on the quality and chemical profiles of final products is pivotal and may benefit future research.

Phytochemical Composition and Protective Effect of Vernonanthura polyanthes Leaf against In Vivo Doxorubicin-Mediated Toxicity.

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (syn.: Vernonia polyanthes Less) is popularly known as "assa-peixe" and its leaves are used in folk medicine mainly to treat respiratory diseases. In this study, we evaluated the cytogenotoxic and anticytogenotoxic potential of the V. polyanthes leaf aqueous extract (VpLAE) and its n-butanol fraction (n-BF) in the presence or absence of doxorubicin (DXR) (pre-, co-, and post-treatments) on a murine model for 24 h or 120 h. The micronucleus test (MN) and the comet assay were used to assess the cytogenotoxic and anticytogenotoxic potential of VpLAE and n-BF (250, 500, and 1000 mg/kg) administered via gavage to Swiss Webster mice. The chemical profiles of VpLAE and n-BF were assessed by liquid chromatography coupled to mass spectrometry, and their metabolites were putatively identified. Lastly, the possible biological activities related to the (anti) cytogenotoxicity of the compounds were predicted using the PASS online webserver. The in vivo results showed that different doses of VpLAE and n-BF did not present cytotoxic activity; however, the MN test revealed a slight mutagenic activity for the 24 h treatments. Moderate genotoxic effects were demonstrated for all treatments in the comet assay. Regarding anticytotoxicity and antimutagenicity, VpLAE and n-BF presented a high cytoprotective potential against DXR toxic effects. In the co-treatment, VpLAE reduced the DXR genotoxicity by ~27%, and n-BF did not demonstrate antigenotoxic potential. In contrast, an antigenotoxic effect was observed for both VpLAE and n-BF in the pre- and post-treatments, reducing DXR genotoxicity by ~41% and ~47%, respectively. Chemical analysis of VpLAE and n-BF showed the presence of eight phenolic compounds, including seven chlorogenic acids and a flavonoid. The PASS online tool predicted antimutagenic, anticancer, antineoplastic, chemoprotective, antioxidant, and radical scavenging activities for all constituents identified in VpLAE and n-BF. V. polyanthes leaves presented a protective effect against DXR cytogenotoxicity. In general, VpLAE and n-BF showed a greater antigenotoxic potential in the pre- and post-treatments. The metabolites putatively identified in VpLAE and n-BF exhibited antioxidant and chemoprotective potential according to computational prediction analysis. Altogether, our results highlight the potential application of V. polyanthes to protect against toxic manifestations induced by DXR.

Plant Metabolites Involved in the Differential Development of a Heliantheae-Specialist Insect.

Balanced nutritional intake is essential to ensure that insects undergo adequate larval development and metamorphosis. Integrative multidisciplinary approaches have contributed valuable insights regarding the ecological and evolutionary outcomes of plant-insect interactions. To address the plant metabolites involved in the larval development of a specialist insect, we investigated the development of Chlosyne lacinia caterpillars fed on Heliantheae species (Tithonia diversifolia, Tridax procumbens and Aldama robusta) leaves and determined the chemical profile of plants and insects using a metabolomic approach. By means of LC-MS and GC-MS combined analyses, 51 metabolites were putatively identified in Heliantheae species and C. lacinia caterpillars and frass; these metabolites included flavonoids, sesquiterpene lactones, monoterpenoids, sesquiterpenoids, diterpenes, triterpenes, oxygenated terpene derivatives, steroids and lipid derivatives. The leading discriminant metabolites were diterpenes, which were detected only in A. robusta leaves and insects that were fed on this plant-based diet. Additionally, caterpillars fed on A. robusta leaves took longer to complete their development to the adult phase and exhibited a greater diapause rate. Hence, we hypothesized that diterpenes may be involved in the differential larval development. Our findings shed light on the plant metabolites that play roles in insect development and metabolism, opening new research avenues for integrative studies of insect nutritional ecology.

Natural Products Diversity in Plant-Insect Interaction between Tithonia diversifolia (Asteraceae) and Chlosyne lacinia (Nymphalidae).

The chemical ecology of plant-insect interactions has been driving our understanding of ecosystem evolution into a more comprehensive context. Chlosyne lacinia (Lepidoptera: Nymphalidae) is an olygophagous insect herbivore, which mainly uses host plants of Heliantheae tribe (Asteraceae). Herein, plant-insect interaction between Tithonia diversifolia (Heliantheae) and Chlosyne lacinia was investigated by means of untargeted LC-MS/MS based metabolomics and molecular networking, which aims to explore its inherent chemical diversity. C. lacinia larvae that were fed with T. diversifolia leaves developed until fifth instar and completed metamorphosis to the adult phase. Sesquiterpene lactones (STL), flavonoids, and lipid derivatives were putatively annotated in T. diversifolia (leaves and non-consumed abaxial surface) and C. lacinia (feces, larvae, pupae, butterflies, and eggs) samples. We found that several furanoheliangolide-type STL that were detected in T. diversifolia were ingested and excreted in their intact form by C. lacinia larvae. Hence, C. lacinia caterpillars may have, over the years, developed tolerance mechanisms for STL throughout effective barriers in their digestive canal. Flavonoid aglycones were mainly found in T. diversifolia samples, while their glycosides were mostly detected in C. lacinia feces, which indicated that the main mechanism for excreting the consumed flavonoids was through their glycosylation. Moreover, lysophospholipids were predominately found in C. lacinia samples, which suggested that they were essential metabolites during pupal and adult stages. These findings provide insights into the natural products diversity of this plant-insect interaction and contribute to uncovering its ecological roles.

Metabolomic analysis applied to chemosystematics and evolution of megadiverse Brazilian Vernonieae (Asteraceae).

Vernonia sensu lato is the largest and most complex genus of the tribe Vernonieae (Asteraceae). The tribe is chemically characterized by the presence of sesquiterpene lactones and flavonoids. Over the years, several taxonomic classifications have been proposed for Vernonia s.l. and for the tribe; however, there has been no consensus among the researches. According to traditional classification, Vernonia s.l. comprises more than 1000 species divided into sections, subsections and series (sensu Bentham). In a more recent classification, these species have been segregated into other genera and some subtribes were proposed, while the genus Vernonia sensu stricto was restricted to 22 species distributed mainly in North America (sensu Robinson). In this study, species from the subtribes Vernoniinae, Lepidaploinae and Rolandrinae were analyzed by UHPLC-UV-HRMS followed by multivariate statistical analysis. Data mining was performed using unsupervised (HCA and PCA) and supervised methods (OPLS-DA). The HCA showed the segregation of the species into four main groups. Comparing the HCA with taxonomical classifications of Vernonieae, we observed that the groups of the dendogram, based on metabolic profiling, were in accordance with the generic classification proposed by Robinson and with previous phylogenetic studies. The species of the genera Stenocephalum, Stilpnopappus, Strophopappus and Rolandra (Group 1) were revealed to be more related to the species of the genus Vernonanthura (Group 2), while the genera Cyrtocymura, Chrysolaena and Echinocoryne (Group 3) were chemically more similar to the genera Lessingianthus and Lepidaploa (Group 4). These findings indicated that the subtribes Vernoniinae and Lepidaploinae are non-chemically homogeneous groups and highlighted the application of untargeted metabolomic tools for taxonomy and as indicators of species evolution. Discriminant compounds for the groups obtained by OPLS-DA were determined. Groups 1 and 2 were characterized by the presence of 3',4'-dimethoxyluteolin, glaucolide A and 8-tigloyloxyglaucolide A. The species of Groups 3 and 4 were characterized by the presence of putative acacetin 7-O-rutinoside and glaucolide B. Therefore, untargeted metabolomic approach combined with multivariate statistical analysis, as proposed herein, allowed the identification of potential chemotaxonomic markers, helping in the taxonomic classifications.