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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, [C 13 /C 14 ]linamarin and [C 13 /C 14 ]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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Research in the field of insect-host plant interactions has indicated that constituents of insect saliva play an important role in digestion and affect host chemical defense responses. However, most efforts have focused on studying the composition and function of regurgitant or saliva produced in the labial glands. Acknowledging the need for understanding the role of the mandibular glands in herbivory, we sought to make a qualitative and semi-quantitative comparison of soluble luminal protein fractions between mandibular and labial glands of Vanessa gonerilla butterfly larvae. Amylase and lysozyme were inspected as possible major enzymatic activities in the mandibular glands aiding in pre-digestion and antimicrobial defense. Although detected, neither of these enzymatic activities was prominent in the luminal protein preparation of a particular type of gland. Proteins isolated from the glands were identified by mass spectrometry and by searching an EST-library database generated for four other nymphalid butterfly species, in addition to the public NCBI database. The identified proteins were also quantified from the data using "Quanty", an in-house program. The proteomic analysis detected chemosensory proteins as the most abundant luminal proteins in the mandibular glands. In comparison to these proteins, the relative amounts of amylase and lysozyme were much lower in both gland types. Therefore, we speculate that the primary role of the mandibular glands in Lepidopteran larvae is chemoreception which may include the detection of microorganisms on plant surfaces, host plant recognition and communication with conspecifics., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.

In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5' and 3' RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5' UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.

Male Idea leuconoe butterflies release a complex mixture of volatiles from their pheromone glands (hairpencils) during courtship. The pheromone components geranyl methyl thioether (2), viridifloric beta-lactone (3) and 6-hydroxy-4-dodecanolide (10) have been synthesized for the first time. Therefore, the structural assignment of these new natural products could be proved. Related 7-hydroxy-5-alkanoides are also present in the extract. The volatiles are embedded in a lipidic, matrix with more than 150 components. This matrix consists of alkanes, alkenes, 2,5-dialkyltetrahydrofurans, secondary alkanols and alkenols as well as alkanones and alkenones. Several regioisomers of the oxidized hydrocarbons occur. The elucidation of double bond positions has been performed by MS using DMDS adducts.