Late-instar monarch caterpillars sabotage milkweed to acquire toxins, not to disarm plant defence.

Sabotaging milkweed by monarch caterpillars (Danaus plexippus) is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow of toxic latex to their feeding site. Here, we show that sabotaging by monarch caterpillars is not only an avoidance strategy. While young caterpillars appear to avoid latex, late-instar caterpillars actively ingest exuding latex, presumably to increase sequestration of cardenolides used for defence against predators. Comparisons with caterpillars of the related but non-sequestering common crow butterfly (Euploea core) revealed three lines of evidence supporting our hypothesis. First, monarch caterpillars sabotage inconsistently and therefore the behaviour is not obligatory to feed on milkweed, whereas sabotaging precedes each feeding event in Euploea caterpillars. Second, monarch caterpillars shift their behaviour from latex avoidance in younger to eager drinking in later stages, whereas Euploea caterpillars consistently avoid latex and spit it out during sabotaging. Third, monarchs reared on detached leaves without latex sequestered more cardenolides when caterpillars imbibed latex offered with a pipette. Thus, we conclude that monarch caterpillars have transformed the ancestral 'sabotage to avoid' strategy into a 'sabotage to consume' strategy, implying a novel behavioural adaptation to increase sequestration of cardenolides for defence.

Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Nectar cardenolides and floral volatiles mediate a specialized wasp pollination system.

Specialization in plant pollination systems can arise from traits that function as filters of flower visitors. This may involve chemical traits such as floral volatiles that selectively attract favoured visitors and non-volatile nectar constituents that selectively deter disfavoured visitors through taste or longer-term toxic effects or both. We explored the functions of floral chemical traits in the African milkweed Gomphocarpus physocarpus, which is pollinated almost exclusively by vespid wasps, despite having nectar that is highly accessible to other insects such as honeybees. We demonstrated that the nectar of wasp-pollinated G. physocarpus contains cardenolides that had greater toxic effects on Apis mellifera honeybees than on Vespula germanica wasps, and also reduced feeding rates by honeybees. Behavioural experiments using natural compositions of nectar compounds showed that these interactions are mediated by non-volatile nectar chemistry. We also identified volatile compounds with acetic acid as a main component in the floral scent of G. physocarpus that elicited electrophysiological responses in wasp antennae. Mixtures of these compounds were behaviourally effective for attraction of V. germanica wasps. The results show the importance of both volatile and non-volatile chemical traits as filters that lead to specialization in plant pollination systems.

Oleandrin enhances radiotherapy sensitivity in lung cancer by inhibiting the ATM/ATR-mediated DNA damage response.

Despite active clinical trials on the use of Oleandrin alone or in combination with other drugs for the treatment of solid tumors, the potential synergistic effect of Oleandrin with radiotherapy remains unknown. This study reveals a new mechanism by which Oleandrin targets ATM and ATR kinase-mediated radiosensitization in lung cancer. Various assays, including clonogenic, Comet, immunofluorescence staining, apoptosis and Cell cycle assays, were conducted to evaluate the impact of oleandrin on radiation-induced double-strand break repair and cell cycle distribution. Western blot analysis was utilized to investigate alterations in signal transduction pathways related to double-strand break repair. The efficacy and toxicity of the combined therapy were assessed in a preclinical xenotransplantation model. Functionally, Oleandrin weakens the DNA damage repair ability and enhances the radiation sensitivity of lung cells. Mechanistically, Oleandrin inhibits ATM and ATR kinase activities, blocking the transmission of ATM-CHK2 and ATR-CHK1 cell cycle checkpoint signaling axes. This accelerates the passage of tumor cells through the G2 phase after radiotherapy, substantially facilitating the rapid entry of large numbers of inadequately repaired cells into mitosis and ultimately triggering mitotic catastrophe. The combined treatment of Oleandrin and radiotherapy demonstrated superior inhibition of tumor proliferation compared to either treatment alone. Our findings highlight Oleandrin as a novel and effective inhibitor of ATM and ATR kinase, offering new possibilities for the development of clinical radiosensitizing adjuvants.

Preliminary investigation of the safe dose of oleandrin when administered orally to Beagles.

OBJECTIVE: The purpose of the study reported herein was to determine the dose of oleander extract and oleandrin (the key pharmacologically active constituent) that could be safely administered PO to dogs. ANIMALS: 42 purebred Beagle dogs were used to study an extract of Nerium oleander. METHODS: 3 studies were performed in 42 purebred young adult (ages 12 months or older) Beagle dogs using a supercritical fluid extract of N oleander leaves. The first study was an 8-day initial dose-ranging study in 2 dogs, a second 7-day repeat-dosing study was performed in 4 dogs, and the final study was performed in 32 dogs where test subjects were given extract or placebo once daily for 28 consecutive days via oral (gavage) administration followed by a 14-day recovery period. RESULTS: At 2.3 µg/kg of oleandrin, there were no observable adverse effects during the duration of the study. Adverse effects were not seen until doses exceeded 6.9 µg/kg of oleandrin, at which time mild, reversible clinical signs were noted. However, a dose > 460 µg of oleandrin/kg was fatal in 1 of 2 dogs in this study. CLINICAL RELEVANCE: The studies reported here, taken in totality, suggest that doses exceeding 6.9 µg/kg of oleandrin may be associated with cardiac abnormalities. An estimated no treatment effective adverse event oral dose of oleandrin appears to be 4.6 µg of oleandrin/kg. Higher doses may be tolerable but should be used with appropriate monitoring.

Chemical constituents of Calotropis procera latex and ultrastructural effects on Haemonchus contortus / Constituintes químicos do látex de Calotropis procera e efeitos ultraestruturais em Haemonchus contortus

Abstract This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.

Resumo Este estudo objetivou avaliar os efeitos anti-helmínticos e ultraestruturais do látex de Calotropis procera sobre Haemonchus contortus. Látex de C. procera foi centrifugado duas vezes à a 10.000xg e dialisado para obter uma fração rica em proteínas, denominada proteínas do látex (LP). E centrifugado e centrifugado a 3.000 rpm, para obter uma fração rica em metabólitos secundários, denominada LNP (látex não proteico). Espécimes de H. contortus expostos à LNP, LP e PBS no Teste de Motilidade dos Nematoides Adultos (TMNA) foram submetidos a microscopia eletrônica de varredura (MEV) e de transmissão (MET), para verificar alterações em sua ultraestrutura. Testes fitoquímicos em LNP indicaram a presença de fenóis, esteroides, alcaloides e cardenolídeos. A presença dos compostos ácido gálico e quercetina em LNP foi caracterizada por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo de proteínas em LP foi de 43,1 ± 1,1 mg/mL e de 7,7 ± 0,3 mg/mL em LNP. No TMNA, LNP e LP inibiram a motilidade de 100% dos nematoides, sendo LNP mais eficaz que LP, e a ivermectina mais eficaz que ambos (p <0,05). Alterações na cutícula de nematoides tratados com LP e LNP foram observadas por MEV e MET. O látex de C. procera apresenta efeito anti-helmíntico sobre H. contortus, causando danos à sua cutícula e outras alterações em sua ultraestrutura.

Improved method for diagnosis of Nerium oleander poisoning in necropsy tissues / Método aprimorado para diagnóstico da intoxicação por Nerium oleander em tecidos de necropsia

Nerium oleander is an ornamental cardiotoxic plant found in tropical and subtropical areas of the World. Its toxicity is related to the content of cardioactive glycosides, mainly oleandrin, found throughout the plant. The present study aimed to describe a new and improved method for oleandrin detection in tissue samples. The determination of oleandrin was made after extraction with a modified QuEChERS technique and measurement by UFLC-MS/MS. A total of 36 guinea pigs (Cavia porcellus) were distributed into 3 groups (n=12): control group that received only water orally (CON), and two treated groups that received hydroalcoholic oleander extract at doses of 150mg.kg-1 (OLE 150) and 300mg.kg-1 (OLE 300) in single oral dose. After three hours, fragments of heart, kidneys, liver and brain were collected for determination of oleandrin levels. The extraction and chromatographic procedures were effective for oleandrin detection and quantification in tissues, with retention time of 1.2 min and detection limit of 0.001μg g-1. The chromatographic analysis of treated guinea pigs indicated that oleandrin is distributed equally among the analyzed tissues. The developed methodology is a reliable, effective and rapid form of diagnosis of N. oleander poisoning based on necropsy tissue samples.(AU)

Nerium oleander é uma planta cardiotóxica ornamental encontrada em áreas tropicais e subtropicais do mundo. Sua toxicidade é relacionada á presença de glicosídeos cardioativos, principalmente a oleandrina, encontrada em toda a planta. O presente estudo objetiva descrever um novo e aprimorado método para detecção da oleandrina em amostras de tecido. A determinação da oleandrina foi feita após extração utilizando técnica modificada de QuEChERS e mensuração por UFLC-MS/MS. Um total de 36 cobaios (Cavia porcellus) foi distribuído em três grupos (n=12): grupo controle que recebeu apenas água por via oral (CON), e dois grupos tratados que receberam extrato hidroalcóolico de oleander nas doses de 150mg.kg-1 (OLE 150) e 300mg.kg-1 (OLE 300) em uma única dose oral. Após três horas, fragmentos do coração, rins, fígado e cérebro foram coletados para determinação dos níveis de oleandrina. A extração e procedimentos cromatográficos foram eficientes na detecção e quantificação da oleandrina nos tecidos, com tempo de retenção de 1,2min e limite de detecção de 0,001μg g-1. A análise cromatográfica dos animais tratados indicou que a oleandrina é distribuída de forma equalizada pelos tecidos analisados. A metodologia desenvolvida representa uma forma de diagnóstica segura, efetiva e rápida da intoxicação por N. oleander a partir de amostras de tecidos de necropsia.(AU)

Estimulación de cardenólidos en brotes de Digitalis purpurea L. cultivados in vitro mediante elicitores / Stimulation of cardenolides production in Digitalis purpurea L. shoot cultures by elicitors addition

Digitalis purpurea L. es una de las principales fuentes de cardenólidos tales como digoxina y digitoxina. Estos fármacos son ampliamente usados en la disfunción cardíaca y para regular las arritmias del corazón. El presente trabajo se realizó con el objetivo de estudiar el efecto de tres elicitores en el cultivo de brotes de Digitalis purpurea var. Roter Berggold para incrementar la producción in vitro de cardenólidos. La elicitación es una estrategia para incrementar la producción de biomasa y metabolitos secundarios en el cultivo in vitro. Los elicitores evaluados fueron ChitoPlant (0,001; 0,01; 0,1 g.L-1); SilioPlant (0,01; 0,1; 1,0 g.L-1) y Jasmonato de metilo (60, 80 y 100 µM), descritos por primera vez para el incremento de cardenólidos. Se demostró que la elicitación es una estrategia viable para el incremento de cardenólidos en brotes de D. purpurea. El ChitoPlant®, redujo la altura sin afectación en el resto de las variables morfológicas evaluadas. Además indujo un incremento significativo en el contenido de cardenólidos. El SilioPlant® (0,01 g.L-1) no provocó afectaciones en la biomasa e incrementó significativamente la síntesis de cardenólidos en los brotes en 3,6 y 6,9 veces el contenido de digoxina y digitoxina respectivamente. La elicitación con el jasmonato de metilo provocó una reducción de la biomasa. Los contenidos de digoxina y digitoxina se incrementaron ligera y significativamente con 80 y 100 µM de jasmonato de metilo respectivamente. El mejor resultado integral se obtuvo con 0,01 g.L-1 de SilioPlant, el cual indujo la mayor producción neta de cardenólidos por frasco de cultivo (4,72 µg digoxina y 88,27 µg digitoxina).

Digitalis purpurea L. is one of the main sources of cardenolides such as digitoxin and digoxin. These drugs are widely used to strengthen cardiac diffusion and to regulate heart rhythm. The aim of this study was to evaluate the influence of three elicitors on shoots of Digitalis purpurea var. Roter Berggold in semisolid media in order to increase cardenolides biosynthesis. Elicitation is a strategy to increase biomass and secondary metabolites production on in vitro cultures. The elicitors evaluated were ChitoPlant (0,001; 0,01; 0,1 g.L-1); SilioPlant (0,01; 0,1; 1,0 g.L-1) and Methyl jasmonate (60, 80, 100 µM), which are reported here to induce cardenolide production for first time. Elicitation resulted an effective strategy to increase cardenolide production on D. purpurea shoot cultures. ChitoPlant induced a decrease in shoots length, but had no effect on the rest of morphological parameters evaluated. As well, ChitoPlant increased cardenolide content. SilioPlant (0,01 g.L-1) did not affect biomass production and at the same time, increased in 3,6-fold and 6,9-fold digoxin and digitoxin content respectively. Elicitation with Methyl jasmonate resulted in decreased biomass production. Digoxin and digitoxin content was slight and significantly increased by Methyl jasmonate 80 and 100 µM respectively. The best integral result was reached with 0,01 g.L-1 of SilioPlant, which induced the highest net yields per culture flask (4,72 µg of digoxin and 88,27 µg of digitoxin).