The scent chemistry of butterflies.

Covering: 1990 up to 2022 Contrary to popular opinion, butterflies exhibit a rich chemistry and elaborate use of volatile compounds, especially for sexual communication, but also for defence. In contrast to night flying moths, in which commonly females are the producers of pheromones, male scent emission is prevalent in butterflies. While visual signals are generally important for long-range attraction, butterfly scent signals are often active only within a short range. Another feature of butterfly scent chemistry is the wide variety of compounds used, including alkaloids, terpenoids, fatty acid derivatives and aromatic compounds, sometimes with unique structures. This contrasts the strucutrally more restricted pheromone chemistry of moths. In this review, the compounds emitted predominately from male butterflies will be discussed and their ecological function explained, if known. The review includes material from 1990 to date, but will also cover older material to provide a necessary background.

Identification and Synthesis of a Macrolide as an Anti-aphrodisiac Pheromone from Males of Heliconius erato phyllis.

Investigation of the contents of the scent glands of the heliconiine butterfly Heliconius erato phyllis via gas chromatography/electroantennography revealed an unprecedented active compound. The males transfer this compound to females during mating. The structure of (2R,6E,10R)-2,6-dimethyl-6-undecen-10-olide, a derivative of geranylacetone, was proposed on the basis of infrared and mass spectrometry spectra and microderivatization and confirmed by racemic and stereoselective syntheses. Bioassays with the synthetic macrolide showed the repellency of this compound, termed phyllisolide, when applied to scent glands of females, identifying it as an anti-aphrodisiac pheromone.

Head and Tail Oxidized Terpenoid Esters from Androconia of Heliconius erato Butterflies.

Heliconius erato is a neotropical butterfly species that is part of a complex mimicry ring, with colorful wing patterns. For intraspecific communication, males use pheromones that are released from two different scent-emitting structures. Scent glands located near the abdominal claspers of males, containing antiaphrodisiac pheromones, release a highly complex mixture of compounds that is transferred to females during mating, rendering them unattractive to other males. On the other hand, androconia, scent-emitting scale areas on the wings of male butterflies, release a structurally more restricted set of compounds that likely serves an aphrodisiac role. We report here on two structurally related compounds that are the major androconial constituents, produced in high amounts and are not volatile due to their high molecular mass. Their structures were established by extensive analysis of mass, infrared, and NMR spectra, as well as microderivatization reactions of the natural extract. After establishing synthetic access, the compounds were unequivocally identified as two unusual head and tail oxidized terpenoids, (4E,8E,12E)-4,8,12-trimethyl-16-oxoheptadeca-4,8,12-trien-1-yl oleate (1) and stearate (2). Although behavioral assays are necessary to fully comprehend their role in the chemical communication of the species, hypotheses for their use by the butterflies are also discussed.

Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-Rich Structural Framework for Monarchs and Swallowtails.

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

Understanding hierarchical spheres-in-grating assembly for bio-inspired colouration.

The vivid iridescent response from particular butterflies is as an excellent example of how micro-engineered hierarchical architectures that combine physical structures and pigmentary inclusions create unique colouration. To date, however, detailed knowledge is missing to replicate such sophisticated structures in a robust, reliable manner. Here, we deliver spheres-in-grating assemblies with colouration effects as found in nature, exploiting embossed polymer gratings and self-assembled light-absorbing micro-spheres.

Anthocyanin complex niosome gel accelerates oral wound healing: In vitro and clinical studies.

An anthocyanin complex (AC), composed of extracts of purple waxy corn and blue butterfly pea petals, and AC niosomes, bilayered vesicles of non-ionic surfactants, were compared in in vitro and clinical studies. Cultured fibroblasts subjected to a scratch wound were monitored for cell viability, cell migration, nuclear morphology and protein expression. Scratched cells showed accelerated wound healing activity, returning to normal 24 h after treatment with AC niosomes (0.002 mg/mL). Western blots and immunocytochemistry indicated upregulation of type I, III and IV collagens, fibronectin and laminins in AC niosome-treated scratched cells. A randomized block placebo-controlled double-blind clinical trial in 60 volunteers (18-60 years old) with oral wounds indicated that AC niosome gel accelerated wound closure, reduced pain due to the oral wounds and improved participants' quality of life more than AC gel, triamcinolone gel and placebo gel. These data are consistent with enhanced delivery of AC to fibroblasts by use of niosomes. AC niosomes activated fibroblasts within wounded regions and accelerated wound healing, indicating that AC niosomes have therapeutic potential.

Development of a Novel Short Synthetic Antibacterial Peptide Derived from the Swallowtail Butterfly Papilio xuthus Larvae.

Insects possess biological defense systems that can effectively combat the invasion of external microorganisms and viruses, thereby supporting their survival in diverse environments. Antimicrobial peptides (AMPs) represent a fast-acting weapon against invading pathogens, including various bacterial or fungal strains. A 37-residue antimicrobial peptide, papiliocin, derived from the swallowtail butterfly Papilio xuthus larvae, showed significant antimicrobial activities against several human pathogenic bacterial and fungal strains. Jelleines, isolated as novel antibacterial peptides from the Royal Jelly (RJ) of bees, exhibit broad-spectrum protection against microbial infections. In this study, we developed a novel antimicrobial peptide, PAJE (RWKIFKKPFKISIHL-NH2), which is a hybrid peptide prepared by combining 1-7 amino acid residues (RWKIFKK-NH2) of papiliocin and 1-8 amino acid residues (PFKISIHL-NH2) of Jelleine-1 to alter length, charge distribution, net charge, volume, amphipaticity, and improve bacterial membrane interactions. This novel peptide exhibited increased hydrophobicity and net positive charge for binding effectively to the negatively charged membrane. PAJE demonstrated antimicrobial activity against both gram-negative and gram-positive bacteria, with very low toxicity to eukaryotic cells and an inexpensive process of synthesis. Collectively, these findings suggest that this novel peptide possesses great potential as an antimicrobial agent.

Photonic effects in natural nanostructures on Morpho cypris and Greta oto butterfly wings.

Photonic crystals are some of the more spectacular realizations that periodic arrays can change the behavior of electromagnetic waves. In nature, so-called structural colors appear in insects and even plants. Some species create beautiful color patterns as part of biological behavior such as reproduction or defense mechanisms as a form of biomimetics. The interaction between light and matter occurs at the surface, producing diffraction, interference and reflectance, and light transmission is possible under suitable conditions. In particular, there are two Colombian butterflies, Morpho cypris and Greta oto, that exhibit iridescence phenomena on their wings, and in this work, we relate these phenomena to the photonic effect. The experimental and theoretical approaches of the optical response visible region were studied to understand the underlying mechanism behind the light-matter interaction on the wings of these Colombian butterflies. Our results can guide the design of novel devices that use iridescence as angular filters or even for cosmetic purposes.

Isolation and analysis of extracellular vesicles in a Morpho butterfly wing-integrated microvortex biochip.

With the function of mediating intercellular communication between cells, extracellular vesicles (EVs) have been intently studied for their physiopathology and clinical application values. However, efficient EV isolation from biological fluids remains a significant challenge. To address this, this work constructs a new microvortex chip that can isolate EVs efficiently by integrating the lipid nanoprobe modified Morpho Menelaus (M. Menelaus) butterfly wing into microfluidic chip. M. Menelaus wing is well known for its orderly arranged periodic nanostructures and can generate microvortex when liquid passes through it, leading to increased interaction between EVs and M. Menelaus wing. In addition, the nanoprobe containing lipid tails can be inserted into EVs through their lipid bilayer membrane structure. Based on the described properties, high-throughput enrichment of EVs with over 70% isolation efficiency was realized. Moreover, it was demonstrated that the nanoprobe system based on M. Menelaus wing enabled downstream biological analysis of nucleic acids and proteins in EVs. Microvortex chips showed potential application value in efficient EV isolation for biomedical research and cancer diagnosis.

The dynamics of cyanide defences in the life cycle of an aposematic butterfly: Biosynthesis versus sequestration.

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.

Health risk associated with the oral consumption of "Chiniy-tref", a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

Chemistry of the Androconial Secretion of the Ithomiine Butterfly Oleria onega.

Ithomiine butterflies use pyrrolizidine alkaloids (PAs) as precursors for male pheromones, such as dihydropyrrolizines or lactones. In contrast to most other ithomiine genera, none of these compounds have ever been detected in Oleria species. The absence of these compounds is thought to be the result of limited access to PA-containing plants. Here we investigate the contents of the androconia of Oleria onega caught in the wild when PA containing plants were abundant. Although the PA lycopsamine was detected in the hairpencils, none of the other known PA-derived compounds were present. Instead, the unsubstituted core of the PA necine base, 1-methylene-1H-pyrrolizine (13), a very unstable compound, was found. The identity of this compound was proven by synthesis. Although its formation in nature appears very likely, 13 is also formed during GC analysis of PAs, making its natural occurrence uncertain. Nevertheless, its reactivity makes it a good candidate for a signaling compound, because its rapid degradation can be used to convey spatial and temporal information. In addition, several other compounds, likely used in intraspecific communication, were identified. All of these compounds are reported for the first time as natural products or from insects. These include 9-hydroxynonanoic acid (21) and (Z)-9-hydroxy-6-enoic acid (18), as well as their condensation products with 11-hexadecenoic- and octadecenoic acids. Furthermore, self-condensation products, such as (Z)-9-[(9-hydroxynon-6-enoyl)oxy]- and 9-[(9-hydroxynonanoyl)oxy]nonanoic acid and non-6-enoic acids (35, 36, 38, 40) were identified, together with the known compounds 2-heptadecanol (39) and 6,10,14-trimethylpentadecan-2-ol (37). In summary, O. onega appears to lack enzymes to produce dihydropyrrolizines. In stark contrast to other ithomiine genera, a unique blend of oxidized fatty acids seems to be used instead.

Differentiation of follicular epithelium in polytrophic ovaries of Pieris napi (Lepidoptera: Pieridae)-how far to Drosophila model.

Lepidoptera together with its sister group Trichoptera belongs to the superorder Amphiesmenoptera, which is closely related to the Antliophora, comprising Diptera, Siphonaptera, and Mecoptera. In the lepidopteran Pieris napi, a representative of the family Pieridae, the ovaries typical of butterflies are polytrophic and consist of structural ovarian units termed ovarioles. Each ovariole is composed of a terminal filament, germarium, vitellarium, and ovariole stalk. The germarium houses developing germ cell clusters and somatic prefollicular and follicular cells. The significantly elongated vitellarium contains linearly arranged ovarian follicles in successive stages of oogenesis (previtellogenesis, vitellogenesis, and choriogenesis). Each follicle consists of an oocyte and seven nurse cells surrounded by follicular epithelium. During oogenesis, follicular cells diversify into five morphologically and functionally distinct subpopulations: (1) main body follicular cells (mbFC), (2) stretched cells (stFC), (3) posterior terminal cells (pFC), (4) centripetal cells (cpFC), and (5) interfollicular stalk cells (IFS). Centripetal cells are migratorily active and finally form the micropyle. Interfollicular stalk cells derive from mbFC as a result of mbFC intercalation. Differentiation and diversification of follicular cells in Pieris significantly differ from those described in Drosophila in the number of subpopulations and their origin and function during oogenesis.

Host specificity pattern and chemical deception in a social parasite of ants.

In natural ecosystems, relationships between organisms are often characterised by high levels of complexity, where vulnerabilities in multi-trophic systems are difficult to identify, yet variation in specific community modules can be traceable. Within the complex community interactions, we can shed new light on dynamics by which co-evolutionary outcomes can inform science-led conservation. Here we assessed host-ant use in six populations of the butterfly Phengaris (=Maculinea) rebeli, an obligate social parasite of Myrmica ants and a model system in evolutionary and conservation ecology. Starting from the initial distribution of eggs, we estimated the survival of the parasite in the wild in nests of seven Myrmica ant species, and analysed the chemical cues evolved by the parasites to subvert its host defences. We found local variations in host specificity that are consistent with similarities found in the chemical profiles of hosts and parasites on different sites. At some sites, only one ant species is successfully exploited; at others, multiple-host populations are used. Understanding how stable or adaptable these associations are is essential knowledge when devising conservation measures to maintain keystone species of ant and locally adapted populations of Phengaris butterfly species, which are rare, threatened and a high priority for conservation worldwide.

Droplet Microarray on Patterned Butterfly Wing Surfaces for Cell Spheroid Culture.

Three-dimensional (3D) cell spheroids have a demonstrated value for in vitro biological research and therapeutics development. Attempts to this technique focus on the development of effective methods for fabricating cell spheroids. Here, inspired by the heterogeneously textured wettability bumps (with hydrophilic peaks and hydrophobic bases) of Stenocara beetle, we present a biotemplated substrate with wettable hydrogel arrays for culturing the cell spheroids. The biotemplates were Morpho butterfly wings with chitin and protein components, which could provide a natural superhydrophobic surface without any modification. The droplet microarrays could be formed for cell spheroid culture on this bioinspired wing substrate by using the hydrogel patterns to hanging droplets. The hanging drop culture method on hydrogel-covered wings has the advantages of high speed, uniform size, and controllable diameter for the formation of 3D cell spheroids. It was demonstrated that drugs produced distinct responses in the 3D cell spheroids compared to conventional two-dimensional cell cultures. As the presented system does not require complex instruments and chemical modifications, our method can simply construct the desired wettability substrates with high biocompatibility for cell culture, drug testing, and other biomedical applications.

Induction of Apoptosis by Pierisin-6 in HPV Positive HeLa and HepG2 Cancer Cells is Mediated by the Caspase-3 Dependent Mitochondrial Pathway.

BACKGROUND: To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines. METHODS: In this study, isolation, and purification of cytotoxic Prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques. Characterization of full-length mRNA of pierisin-6 gene was performed using 3'/5' RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of Pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide, and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS. RESULTS: In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from the mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac- DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation. CONCLUSION: Pierisin-6 represents a promising therapeutic approach for liver cancer patients.

Analysis of butterfly reproductive proteins using capillary electrophoresis and mass spectrometry.

A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000-6000) and a higher m/z range (6000-12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.

An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi.

A method for off-line CE-MALDI-TOF-MS and MS2, and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS2 spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.

Comparative study on nanostructured order-disorder in the wing eyespots of the giant owl butterfly, Caligo memnon.

A characteristic feature of the giant owl butterfly, i.e. Caligo memnon, is its big wing eyespot. This feature could serve as deceiving functionality for the butterfly against predators. As evidenced by scanning electron microscope (SEM) image on black part of eyespot, the scales on wing eyespot contain nanostructured ridges and cross-ribs. Applying direct measurement, statistical method, and Fourier analysis, the authors evidence that these nanostructures display order-disorder in their shape and position. The autocorrelation of SEM image provides average values of characteristic periods of the order-disorder nanostructures together with an estimation of corresponding correlation lengths. Linecuts obtained from the Fourier transform of SEM image were also analysed with the Hosemann function to extract similar information. These analyses indicate that the nanostructured order-disorder may contribute to blackness on wing eyespot. The authors thus conclude that the blackness on wing eyespot of C. memnon could be attributed to contributions from both the nanostructured order-disorder and melanin pigment.