Male ithomiine butterflies (Nymphalidae: Danainae) have hairpencils on the forewings (i.e., androconia) that disseminate semiochemicals during courtship. While most ithomiines are known to contain derivatives of pyrrolizidine alkaloids, dihydropyrrolizines, or γ-lactones in these androconia, here we report on a new class of fatty acid esters identified in two subspecies, Ithomia salapia aquinia and I. s. derasa. The major components were identified as isoprenyl (3-methyl-3-butenyl) (Z)-3-acetoxy-11-octadecenoate, isoprenyl (Z)-3-acetoxy-13-octadecenoate (12) and isoprenyl 3-acetoxyoctadecanoate (11) by GC/MS and GC/IR analyses, microderivatizations, and synthesis of representative compounds. The absolute configuration of 12 was determined to be R. The two subspecies differed not only in the composition of the ester bouquet, but also in the composition of more volatile androconial constituents. While some individuals of I. s. aquinia contained ithomiolide A (3), a pyrrolizidine alkaloid derived γ-lactone, I. s. derasa carried the sesquiterpene α-elemol (8) in the androconia. These differences might be important for the reproductive isolation of the two subspecies, in line with previously reported low gene exchange between the two species in regions where they co-occur. Furthermore, the occurrence of positional isomers of unsaturated fatty acid derivatives indicates activity of two different desaturases within these butterflies, Δ9 and Δ11, which has not been reported before in male Lepidoptera.

Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography.

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.

Neotropical Heliconius butterflies are members of various mimicry rings characterized by diverse colour patterns. In the present study we investigated whether a similar diversity is observed in the chemistry of volatile compounds present in male wing androconia. Recent research has shown that these androconia are used during courting of females. Three to five wild-caught male Heliconius individuals of 17 species and subspecies were analyzed by GC/MS. Most of the identified compounds originate from common fatty acids precursors, including aldehydes, alcohols, acetates or esters preferentially with a C18 and C20 chain, together with some alkanes. The compounds occurred in species-specific mixtures or signatures. For example, octadecanal is characteristic for H. melpomene, but variation in composition between the individuals was observed. Cluster analysis of compound occurrence in individual bouquets and analyses based on biosynthetic motifs such as functional group, chain length, or basic carbon-backbone modification were used to reveal structural patterns. Mimetic pairs contain different scent bouquets, but also some compounds in common, whereas sympatric species, both mimetic and non-mimetic, have more distinct compound compositions. The compounds identified here may play a role in mate choice thus helping maintain species integrity in a butterfly genus characterized by pervasive interspecific gene flow.

Macrolides are a relatively common structural motif prevalent in Nature. However, the structures of these large ring lactones have been relatively difficult to elucidate via NMR spectroscopy due to the minute amounts of compounds that are sometimes obtainable from natural sources. Thus, GC-MS analysis of individual macrolactones has become the method of choice for the structural identification of these compounds. Here we discuss the mass spectrometric behavior of aliphatic macrolides, evaluating spectra from numerous compounds of various ring size, including derivatives containing methyl branches as well as double bonds. The specific fragmentation of these macrolactones under electron impact conditions allows for the development of a general rule set aimed at the identification of similar compounds by mass spectrometry. In addition, the mass spectra of dimethyl disulfide adducts of unsaturated macrolides are discussed. The mass spectra of almost 50 macrolides are presented.

Sex specific pheromones are known to play an important role in butterfly courtship, and may influence both individual reproductive success and reproductive isolation between species. Extensive ecological, behavioural and genetic studies of Heliconius butterflies have made a substantial contribution to our understanding of speciation. Male pheromones, although long suspected to play an important role, have received relatively little attention in this genus. Here, we combine morphological, chemical, and behavioural analyses of male pheromones in the Neotropical butterfly Heliconius melpomene. First, we identify putative androconia that are specialized brush-like scales that lie within the shiny grey region of the male hindwing. We then describe putative male sex pheromone compounds, which are largely confined to the androconial region of the hindwing of mature males, but are absent in immature males and females. Finally, behavioural choice experiments reveal that females of H. melpomene , H. erato and H. timareta strongly discriminate against conspecific males which have their androconial region experimentally blocked. As well as demonstrating the importance of chemical signalling for female mate choice in Heliconius butterflies, the results describe structures involved in release of the pheromone and a list of potential male sex pheromone compounds.

Twenty-four strains of marine Roseobacter clade bacteria were isolated from macroalgae and investigated for the production of quorum-sensing autoinducers, N-acylhomoserine lactones (AHLs). GC/MS analysis of the extracellular metabolites allowed us to evaluate the release of other small molecules as well. Nineteen strains produced AHLs, ranging from 3-OH-C10: 0-HSL (homoserine lactone) to (2E, 11Z)-C18: 2-HSL, but no specific phylogenetic or ecological pattern of individual AHL occurrence was observed when cluster analysis was performed. Other identified compounds included indole, tropone, methyl esters of oligomers of 3-hydroxybutyric acid, and various amides, such as N-9-hexadecenoylalanine methyl ester (9C16: 1-NAME), a structural analogue of AHLs. Several compounds were tested for their antibacterial and antialgal activity on marine isolates likely to occur in the habitat of the macroalgae. Both AHLs and 9-C16: 1-NAME showed high antialgal activity against Skeletonema costatum, whereas their antibacterial activity was low.

A new arabinomannan has been isolated from the cell wall of the green algae Chlorella vulgaris by extraction with 0.1M NaOH, dialysis and SEC fractionation. Mw was about 8000u. Terminal, 2- and 5-O-linked arabinofuranosyl residues, and 2,6-O-linked mannopyranosyl residues were detected as the main constituents beside some minor mannose components by methylation analysis. Electrospray ionisation mass spectrometry (ESI–MS) with collision induced dissociation (CID) up to MS3 experiments of oligosaccharides obtained by partial methanolysis or partial acid hydrolysis indicated the presence of Man→Man- and Ara→Ara- as well as Ara→Man- and also Man→Ara-sequences. MS2 experiments gave evidence of a (1→6)-linked mannan, probably also including some (1→2)-linked mannosyl and 5-linked arabinofuranosyl residues. Mannooligosaccharides up to DP5 with mainly (1→6)-, but at higher DP also (1→2)-linkages were obtained by acid hydrolysis, and arabino oligomers up to DP4 could be detected after mild methanolysis. In accordance with results from methylation analysis and ESI–MS/CID t-β-Araf, 2-α-Araf, 5-α-Araf and 2,6-α-Manp were identified from homo- and heteronuclear 1D- and 2D NMR experiments in a molar ratio of ∼2:2:1:2. A highly branched structure is suggested with a 6- and 2-O-linked mannan main chain, comprising 5-Araf residues. Araf-β-[(1→2)-Araf-α-(1→]n side chains with an average chain length of 2 are linked to the mannan main chain. The configuration of Ara is d. The new polysaccharide shows structural similarities with the (lipo)arabinomannans of Mycobacterium tuberculosis.