Pyrrolizidine alkaloids in tiger moths: trends and knowledge gaps

Introduction A curious relationship exists between a group of plants, the pyrrolizidine alkaloids they contain, and tiger moths of the family Arctiidae. Tiger moths possess an impressive array of chemicals, either produced de novo or sequestered from plants, that protect them to a greater or lesser degree from predators and parasites. These chemicals include cyossin (Teas et al. , 1966), biogenic amines (Bisset et al. , 1959, 1960; Rothschild and Aplin, 1971), pyrazines (Rothschild et al. , 1984), polyphenolics (Hesbacher et al. , 1995), iridoid glycosides (Bowers and Stamp, 1997), and cardenolides (Rothschild et al. , 1970, 1973; Wink and von Nickisch-Rosenegk, 1997); however, no group of compounds, it seems, has influenced the natural history and behavior of tiger moths as the pyrrolizidine alkaloids (PAs) have done (Weller et al. , 2000a). Several excellent reviews have been written about these compounds from the perspective of their chemistry, the plants that produce them (Bull et al. ; 1968; Mattocks, 1986; Hartmann and Witte, 1995), and the insects that utilize them (Schneider, 1986; Boppre, 1990; Hartmann and Ober, 2000), but none has focussed exclusively, on their intimate relationships with tiger moths. The members of the family Arctiidae, which numbers over 11 000 species, are often brilliantly colored (Watson and Goodger, 1986; Holloway, 1988; Weller et al. , 2000a). In addition to standard aposematic red, yellow, or black patterns, adults and larvae may have iridescent blue and green, or even pearly white, coloration. White can be considered aposematic when individuals rest conspicuously on green vegetation.

Adult tiger moths exhibit a wide range of palatabilities to the insectivorous big brown bat Eptesicus fuscus. Much of this variation is due to plant allelochemics ingested and sequestered from their larval food. By using a comparative approach involving 15 species from six tribes and two subfamilies of the Arctiidae we have shown that tiger moths feeding on cardiac glycoside-containing plants often contain highly effective natural feeding deterrents. Feeding on pyrrolizidine alkaloid-containing plants is also an effective deterrent to predation by bats but less so than feeding on plants rich in cardiac glycosides. Moths feeding on plants containing iridoid glycosides and/or moths likely to contain biogenic amines were the least deterrent. By manipulating the diet of several tiger moth species we were able to adjust their degree of palatability and link it to the levels of cardiac glycosides or pyrrolizidine alkaloids in their food. We argue that intense selective pressure provided by vertebrate predators including bats has driven the tiger moths to sequester more and more potent deterrents against them and to acquire a suite of morphology characteristics and behaviors that advertise their noxious taste.

The focus of this study was to reconstruct a phylogenetic hypothesis for the moth subfamily Arctiinae (tiger moths, woolly bears) to investigate the evolution of larval and adult pharmacophagy of pyrrolizidine alkaloids (PAs) and the pathway to PA chemical specialization in Arctiinae. Pharmacophagy, collection of chemicals for non-nutritive purposes, is well documented in many species, including the model species Utetheisa ornatrix L. A total of 86 exemplar ingroup species representing tiger moth tribes and subtribes (68 genera) and nine outgroup species were selected. Ingroup species included the most species-rich generic groups to represent the diversity of host-plant associations and pharmacophagous behaviors found throughout Arctiinae. Up to nine genetic markers were sequenced: one mitochondrial (COI barcode region), one nuclear rRNA (D2 region, 28S rRNA), and seven nuclear protein-coding gene fragments: elongation factor 1-α protein, wingless, ribosomal protein subunit S5, carbamoylphosphate synthase domain regions, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and cytosolic malate dehydrogenase. A total of 6984 bp was obtained for most species. These data were analyzed using model-based phylogenetic methods: maximum likelihood (ML) and Bayesian inference (BI). Ancestral pharmacophagous behaviors and obligate PA associations were reconstructed using the resulting Bayes topology and Reconstructing Ancestral States in Phylogenies (RASP) software. Our results corroborate earlier studies on the evolution of adult pharmacophagous behaviors, suggesting that this behavior arose multiple times and is concentrated in the phaegopterine-euchromiine-ctenuchine clade (PEC). Our results suggest that PA specialization may have arisen early in the phylogeny of the subfamily and that facultative larval pharmacophagous behaviors are the derived condition.

The evolution of chemical defences and mating systems in tiger moths (Lepidoptera: Arctiidae)

A preliminary phylogeny for the Callimorphini and related tribes was constructed based on adult morphology. The data matrix, consisting of 91 taxa and 116 characters (299 states), was analyzed using maximium parsimony. To test the monophyly of the Callimorphini s.s. and determine the placement of the Euchaetes group, 49 ingroup species and 42 outgroup species were included. Callimorphini was represented by 23 species (11 genera), and the Euchaetes group was represented by 26 species (6 genera). Outgroup taxa include both root genera from the Arctiini (10 genera, 12 species) and representative genera from related tribes as “internal outgroups.” Internal outgroups included representatives from Phaegopterini (6 genera, 9 species), Nyctemerini (1 genus, 9 species), and Pericopini (7 genera, 12 species). The data matrix included both nongenitalic and genitalic characters as follows: 7 head (17 states), 5 leg (10 states), 22 wing (57 states), 12 thoracic (29 states), 39 male abdominal and genitalic characters (95 states) and 31 female abdominal and genitalia (91 states). Of these, 36 were multistate and were treated as unordered. Heuristic searches with 20, 100, and 1000 random–taxon additions were performed to sample multiple tree islands. This study also assessed the utility of adult genitalic character systems for confident resolution of older divergences. The resulting cladistic analysis demonstrated that phylogenetic relationships among closely related genera were recovered; however, genitalic characters do not fully replace information from immature stages. Relationships among major clades remained poorly supported as determined by taxon and character jackknifes. Based on these results, a revised concept for Callimorphini is proposed based on well–supported relationships. A checklist of callimorphine genera also is provided. The Old World genus Nyctemera is placed in the Pericopini rev. placement and Euchaetes and allied genera are placed in the Phaegopterini rev. placement.KEY WORDS: Arctiidae, Callimorphini, phylogenyCallimorphini (Lepidoptera: Arctiidae: Arctiinae), like many tiger moths, are cosmopolitan in distribution and most diverse in the tropics. Adults frequently are brightly colored with orange, pink, yellow, and red scales on the wings and abdomen (Ferguson 1985, Holloway et al. 2001). Some members of the Callimorphini, a small tribe of approximately 108 species, resemble butterflies in that they are diurnal (e.g., Utetheisa Hübner) or have large wings and “slender” bodies characteristic of butterflies (e.g., Haploa Hübner, Callimorpha Latreille) (Figs. 1, 2). Callimorphini, and tiger moths more generally, have captured the attention of scientists because of their coloration and their intriguing biology. In general, arctiid larvae and adults defend themselves from predators with biogenic amines, often in combination with host–plant derived chemicals such as pyrrolizidine alkaloids (PAs), cardiac glycosides (CGs), or sesquiterpenes (reviews: Weller et al. 1999; Conner & Weller 2004). These compounds can be found in all life stages in several species. Callimorphini is pivotal for understanding the evolutionary transitions among the different defense strategies within the subfamily Arctiinae (Fig. 3; Weller et al. 1999). Most callimorphines use pyrrolizidine

Development and validation of a QuEChERS method coupled to liquid chromatography and high resolution mass spectrometry to determine pyrrolizidine and tropane alkaloids in honey

Chapter 1 17 extrapolation. T10 values are obtained by multiplying T25 values by 10/25. At the current state of the art, the T25 with a MOE cut-off value of 25000 and the T10 with a MOE cut-off value of 10000 (also applied when using the BMDL10) are used when a BMDL10 is not available. A value of 10000 incorporates three uncertainty factors including a factor 100 for species differences and human variability in kinetics and dynamics, a factor of 10 for the variability in cell cycle control and DNA repair within humans, and a factor of 10 because the BMDL10 is not a no observed adverse effect level (NOAEL) (EFSA, 2005).

Since 1890, many observations of danaine butterflies visiting dry plants of several families in the Old and New World tropics have been published. For 50 years, it has been recognised that Danainae, along with various other insects, seek out 1,2-dehydropyrrolizidine ester alkaloids (PAs) independently of and in addition to their nutritive requirements and utilise them to increase their chances for survival and biological fitness. This represents an unusual type of insect-plant relationship (“PA-pharmacophagy”), with remarkable peculiarities but also with gaps in knowledge, many of which can be filled by employing PA-baiting. We review and analyse the history of records on the attraction of adult insects to PAs and unveil the complex background information on PA-chemistry, PA-producing plants (“PA-plants”), and PA-sequestering insects (“PA-insects”) in order to come up with practical tips for successful baiting with PAs (“PA-baiting”). Studying PA-pharmacophagy integrates taxonomy, behaviour, and ecology with evolutionary biology, chemistry, and toxicology. With basic knowledge of PA-chemistry and bearing the general peculiarities of PA-plants and PA-insects in mind, PA-baiting can be conducted easily and successfully to address many questions on the multifaceted ecology of pyrrolizidine alkaloids. We aim to encourage field researchers in the tropics to employ PA-baiting as a valuable research method in this field of integrative biology.

Aposematic organisms warn predators of their unprofitability using a combination of defenses, including visual warning signals, startling sounds, noxious odors, or aversive tastes. Using multiple lines of defense can help prey avoid predators by stimulating multiple senses and/or by acting at different stages of predation. We tested the efficacy of three lines of defense (color, smell, taste) during the predation sequence of aposematic wood tiger moths (Arctia plantaginis) using blue tit (Cyanistes caeruleus) predators. Moths with two hindwing phenotypes (genotypes: WW/Wy = white, yy = yellow) were manipulated to have defense fluid with aversive smell (methoxypyrazines), body tissues with aversive taste (pyrrolizidine alkaloids) or both. In early predation stages, moth color and smell had additive effects on bird approach latency and dropping the prey, with the strongest effect for moths of the white morph with defense fluids. Pyrrolizidine alkaloid sequestration was detrimental in early attack stages, suggesting a trade-off between pyrrolizidine alkaloid sequestration and investment in other defenses. In addition, pyrrolizidine alkaloid taste alone did not deter bird predators. Birds could only effectively discriminate toxic moths from non-toxic moths when neck fluids containing methoxypyrazines were present, at which point they abandoned attack at the consumption stage. As a result, moths of the white morph with an aversive methoxypyrazine smell and moths in the treatment with both chemical defenses had the greatest chance of survival. We suggest that methoxypyrazines act as context setting signals for warning colors and as attention alerting or “go-slow” signals for distasteful toxins, thereby mediating the relationship between warning signal and toxicity. Furthermore, we found that moths that were heterozygous for hindwing coloration had more effective defense fluids compared to other genotypes in terms of delaying approach and reducing the latency to drop the moth, suggesting a genetic link between coloration and defense that could help to explain the color polymorphism. Conclusively, these results indicate that color, smell, and taste constitute a multimodal warning signal that impedes predator attack and improves prey survival. This work highlights the importance of understanding the separate roles of color, smell and taste through the predation sequence and also within-species variation in chemical defenses.

In Indonesia, the market demand for herbal products keeps growing, and as a result, herbal products increasingly provide economic and perceived clinical benefits. A risk and benefit assessment are crucial to be performed to support the safe use of herbal products although the consumers perceive herbal product as “safe” and “natural” and thus “healthy”. The aim of the PHD thesis was to perform an assessment of potential risks and some benefits of herbal products available in the Indonesian market. The model compounds chosen included especially naturally occurring genotoxic and carcinogenic botanical constituents including alkenylbenzenes (ABs) and pyrrolizidine alkaloids (PAs). Beneficial effects focussed on potential PPARg activation by the carotenoids bixin and crocetin. Existing but also novel testing strategies were used to evaluate the relevance of effects at estimated human intake levels. Altogether, it can be concluded that the risk assessment using the Margin of Exposure approach combined with Haber’s rule can be used to prioritize risk management actions to prevent the adverse health effects of consuming Indonesian herbal products containing genotoxic carcinogens. In addition, a novel testing strategy, combining in vitro and PBK modeling-facilitated reverse dosimetry was found to facilitate risk and benefit assessment of botanical compounds without the need for animal experiments and/or human intervention studies. It is important to note that this conclusion holds for herbal products collected by targeted sampling, and not for all herbal products on the Indonesian market. Many aspects, including variability in detected levels of the targeted compounds, variability in recommended daily use mentioned on the label, interindividual variation of exposure among Indonesian people, absence of a generally accepted method to take shorter-than-lifetime exposure into account, the knowledge gaps in modes of action, selection of the best in vitro model for QIVIVE, potential combination effects, the chemical-specific parameters needed for PBK modeling and availability of in vivo studies to validate the predictions should be considered for future research. Seven actions including (1) applying Good Agricultural and Collection Practices (GACP) for farmers, (2) applying good manufacturing practice (GMP) of herbal product and food safety training for manufacturers and producers, (3) development of a toxicity database of medicinal botanicals used in Indonesia, (4) restriction of the exposure to genotoxic carcinogenic compounds by establishing MPLs and refining the label requirements for botanicals and botanical preparations, (5) use of human biomonitoring (HBM) and PBK modeling for a more refined exposure, risk and benefit analysis of Indonesian herbal products, (6) incorporation of Chemical Specific Adjustment Factors (CSAFs) for interspecies and interindividual variation in kinetics within the human population in the risk assessment, and (7) exploring the beneficial effects of botanicals and botanical preparations, were proposed to improve safety and efficacy of botanicals and botanical preparations on the Indonesian market.

General background: Sesbania grandiflora (L.) Pers., widely known as white turi, is traditionally used as analgesic, laxative, and other medicinal purposes. Specific background: Despite its therapeutic benefits, the plant contains secondary metabolites, including triterpenoids and alkaloids, with potential hepatotoxic properties, particularly pyrrolizidine alkaloids. Knowledge gap: Previous studies have indicated general toxicity of turi extracts but limited evidence exists on its subacute hepatic toxicity measured through enzymatic markers in animal models. Aim: This study investigated the toxicity of white turi leaf extract on liver function by assessing SGOT and SGPT levels in mice (Mus musculus). Results: Laboratory experiments using a posttest-only control group design were conducted with multiple dosage levels. Statistical analysis with Kruskal-Wallis revealed no significant differences in SGOT (p = 0.896) and SGPT (p = 0.527) levels between treatment and control groups, while ANOVA showed no significant change in liver weight (p = 0.089). Novelty: This research provides the first evidence that ethanolic white turi leaf extract, within the tested doses and 14-day observation, does not induce measurable hepatic dysfunction in mice. Implications: The findings suggest relative safety of white turi extract at tested doses, supporting its potential traditional use while highlighting the importance of further chronic toxicity studies. Highlights : White turi leaf extract showed no hepatotoxic effect in mice. SGOT-SGPT levels and liver weight remained within normal range. Supports safe traditional use but requires chronic toxicity evaluation. Keywords : Sesbania Grandiflora, White Turi Leaf, SGOT, SGPT, Hepatotoxicity

Presence and distribution of sensory structures on the mouthparts of self-medicating moths

The tiger moth caterpillars Grammia geneura and Estigmene acrea defend themselves against parasites by feeding on plants containing phytochemicals toxic to the parasites: E. acrea eats and sequesters pyrrolizidine alkaloids; G. geneura eats and sequesters iridoid glycosides as well. The caterpillars detect these compounds by means of specialized chemoreceptor gustatory cells that stimulate feeding behavior. Bernays and Singer showed that, compared with control caterpillars, the increase in taste cell firing rate in response to the iridoid catalpol and the pyrrolizidine alkaloid seneciphylline N -oxide was greater in parasitized G. geneura . In contrast, the response of a gustatory cell sensitive to the feeding deterrent caffeine was decreased. There was no difference in the response to sucrose, which is detected through the same gustatory cells as iridoids, indicating that the change affected the iridoid receptor or the subsequent signaling pathway rather than being cell-wide. Similarly, gustatory cells in parasitized E. acrea showed an enhanced response to seneciphylline N -oxide compared with unparasitized caterpillars, a reduced response to the feeding deterrent protocatechuic acid, and an inconsistent response to sucrose. Thus, parasitization appears to alter the sensitivity of caterpillar gustatory cells to promote the ingestion of substances toxic to the parasites and to inhibit the response to substances that inhibit feeding behavior. E. A. Bernays, M. S. Singer, Insect defences: Taste alteration and endoparasites. Nature 436 , 476 (2005). [PubMed]

ABSTRACT. Despite decades of research on Ranchman's tiger moth (Platyprepia virginalis), little is known about the behavior and ecology of the adult life stage. To address this knowledge gap, we conducted surveys to quantify the spatial distribution of moths, and conducted laboratory and field oviposition assays as well as a field oviposition survey. We found that P. virginalis exhibits hilltopping behavior, a mate-locating strategy where individuals congregate on hilltops to increase the likelihood of sexual encounters. This behavior is common across many insect orders, but there are few examples of moths exhibiting this behavior. We found no evidence supporting our hypothesis that bush lupine (Lupinus arboreus), the primary larval hostplant within our study site, is the preferred oviposition hostplant. The opportunistic discovery of egg clutches on seaside daisy plants (Erigeron glaucus) led us to conduct a no-choice larval feeding assay to determine its suitability as a hostplant. We found that larvae ...

These days, a growing consumer demand and scientific interest can be observed for nutraceuticals of natural origin, including apiculture products. Due to the growing emphasis on environmental protection, extensive research has been conducted on the pesticide and heavy metal contamination of bee products; however, less attention is devoted on other food safety aspects. In our review, scientific information on the less-researched food safety hazards of honey, bee bread, royal jelly, propolis, and beeswax are summarized. Bee products originating from certain plants may inherently contain phytotoxins, like pyrrolizidine alkaloids, tropane alkaloids, matrine alkaloids, grayanotoxins, gelsemium alkaloids, or tutin. Several case studies evidence that bee products can induce allergic responses to sensitive individuals, varying from mild to severe symptoms, including the potentially lethal anaphylaxis. Exposure to high temperature or long storage may lead to the formation of the potentially toxic 5-hydroxymethylfurfural. Persistent organic pollutants, radionuclides, and microplastics can potentially be transferred to bee products from contaminated environmental sources. And lastly, inappropriate beekeeping practices can lead to the contamination of beekeeping products with harmful microorganisms and mycotoxins. Our review demonstrates the necessity of applying good beekeeping practices in order to protect honeybees and consumers of their products. An important aim of our work is to identify key knowledge gaps regarding the food safety of apiculture products.