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Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown., (© 2024. The Author(s).)

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 C 18 and C 20 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.

Many insect species sequester compounds acquired from their host plants for defense against natural enemies. The distribution of these compounds is likely to affect both their efficacy as defenses, and their costs. In this study we examined the distribution of sequestered iridoid glycosides (IGs) in two congeneric species of nymphalid butterfly, Euphydryas anicia and E. phaeton, and found that the pattern of localization of IGs differed between the two species. Although IG concentrations were quite high in the heads of both species, the relative concentrations in wings and abdomens differed substantially. Euphydryas anicia had relatively high IG concentrations in their abdomens and low IG concentrations in their wings, whereas the reverse was true in E. phaeton. We interpret these results in light of two current hypotheses regarding where sequestered chemicals should be localized: that they should be found in wings, which would allow non-lethal sampling by predators; and that their distribution is constrained by the distribution of tissue types to which sequestered compounds bind. We also offer the third hypothesis, that costs of storage may differ among body parts, and that the localization of compounds may reflect a cost-reduction strategy. Results from E. phaeton were consistent with all three of these non-mutually exclusive hypotheses, whereas results from E. anicia were only consistent with the notion that tissue bias among body parts plays a role in IG distribution. The finding that these two congeneric butterflies exhibit different patterns of IG localization suggests that they have been shaped by different selection regimes.

Gas chromatography (GC) and mass spectrometry (MS) were used to determine the stereoisomeric compositions of 6,10,14-trimethylpentadecan-2-ol and 6,10,14-trimethylpentadecan-2-one in wing extracts from 17 Bicyclus butterfly species from different regions of Africa. All samples were purified using solid phase extraction (SPE). Since some species contained both alcohol and ketone, these were separated and the ketone was reduced to the alcohol before analysis as either (R)-trans-chrysanthemoyl or (S)-2-acetoxypropionyl esters. A novel asymmetric synthesis was developed for a reference mixture of (2R/S,6S,10R)-6,10,14-trimethylpentadecan-2-ol with known composition of the eight stereoisomers. The mixture then was used as the (R)-trans-chrysanthemoyl esters to correlate each of the eight gas chromatographic peaks to a specific stereoisomer of the extracted wing compounds. Seven butterfly species showed (2R,6R,10R)-configuration of the alcohol, four species contained minute amounts of alcohol too small to determine the stereochemistry, nine species showed (6R,10R)-configuration of the ketone, and one species contained minute amounts of ketone too small to determine the stereochemistry. No other stereoisomers of alcohol or ketone could be detected in the extracts, and the quantities of the compounds in the wing extracts varied from 5 to 900 ng per sample for each species.

Changes in dietary preferences in animal species play a pivotal role in niche specialization. Here, we investigate how divergence of foraging behaviour affects the trophic position of animals and thereby their role for ecosystem processes. As a model, we used two closely related bat species, Myotis myotis and M. blythii oxygnathus, that are morphologically very similar and share the same roosts, but show clear behavioural divergence in habitat selection and foraging. Based on previous dietary studies on synanthropic populations in Central Europe, we hypothesised that M. myotis would mainly prey on predatory arthropods (i.e., secondary consumers) while M. blythii oxygnathus would eat herbivorous insects (i.e., primary consumers). We thus expected that the sibling bats would be at different trophic levels. We first conducted a validation experiment with captive bats in the laboratory and measured isotopic discrimination, i.e., the stepwise enrichment of heavy in relation to light isotopes between consumer and diet, in insectivorous bats for the first time. We then tested our trophic level hypothesis in the field at an ancient site of natural coexistence for the two species (Bulgaria, south-eastern Europe) using stable isotope analyses. As predicted, secondary consumer arthropods (carabid beetles; Coleoptera) were more enriched in (15)N than primary consumer arthropods (tettigoniids; Orthoptera), and accordingly wing tissue of M. myotis was more enriched in (15)N than tissue of M. blythii oxygnathus. According to a Bayesian mixing model, M. blythii oxygnathus indeed fed almost exclusively on primary consumers (98%), while M. myotis ate a mix of secondary (50%), but also, and to a considerable extent, primary consumers (50%). Our study highlights that morphologically almost identical, sympatric sibling species may forage at divergent trophic levels, and, thus may have different effects on ecosystem processes.

Omnivorous animals feed on several food items that often differ in macronutrient and isotopic composition. Macronutrients can be used for either metabolism or body tissue synthesis and, therefore, stable C isotope ratios of exhaled breath (delta(13)C(breath)) and tissue may differ. To study nutrient routing in omnivorous animals, we measured delta(13)C(breath) in 20-g Carollia perspicillata that either ate an isotopically homogeneous carbohydrate diet or an isotopically heterogeneous protein-carbohydrate mixture. The delta(13)C(breath) converged to the delta(13)C of the ingested carbohydrates irrespective of whether proteins had been added or not. On average, delta(13)C(breath) was depleted in (13)C by only ca. -2 per thousand in relation to the delta(13)C of the dietary carbohydrates and was enriched by +8.2 per thousand in relation to the dietary proteins, suggesting that C. perspicillata may have routed most ingested proteins to body synthesis and not to metabolism. We next compared the delta(13)C(breath) with that of wing tissue (delta(13)C(tissue)) in 12 free-ranging, mostly omnivorous phyllostomid bat species. We predicted that species with a more insect biased diet--as indicated by the N isotope ratio in wing membrane tissue (delta(15)N(tissue))--should have higher delta(13)C(tissue) than delta(13)C(breath) values, since we expected body tissue to stem mostly from insect proteins and exhaled CO(2) to stem from the combustion of fruit carbohydrates. Accordingly, delta(13)C(tissue) and delta(13)C(breath) should be more similar in species that feed predominantly on plant products. The species-specific differences between delta(13)C(tissue) and delta(13)C(breath) increased with increasing delta(15)N(tissue), i.e. species with a plant-dominated diet had similar delta(13)C(tissue) and delta(13)C(breath) values, whereas species feeding at a higher trophic level had higher delta(13)C(tissue) than delta(13)C(breath) values. Our study shows that delta(13)C(breath) reflect the isotope ratio of ingested carbohydrates, whereas delta(13)C of body tissue reflect the isotope ratio of ingested proteins, namely insects, supporting the idea of isotopic routing in omnivorous animals.

In 1997 and 1998 we weighed nestling tree swallows (Tachycineta bicolor) and measured selected body components at two colonies: Acadia National Park on Mt. Desert Island, and at Orono, ME. We used differences in mean growth variables among individual nestlings to evaluate differences between colonies, years, and amount of total mercury (THg) in carcasses and methyl mercury (MeHg) in feathers. We marked nestlings on the day hatched and measured body components every day in 1997 and every other day in 1998 until nestlings fledged. We calculated linear growth rates and asymptotic means as appropriate. In 1998, linear growth rate of weight was higher at Acadia than at Orono, but not different in 1997. We detected no mean differences in asymptotic mean weight of nestlings between colonies or years. In 1997, mean linear growth rates of the wing (chord), tail, tarsus, and mandible were higher at Acadia than at Orono. The amount of MeHg in feathers was associated with a lower linear growth rate of weight during early age (2-10 days), but asymptotic mean weight during days 11-16 was not different. No effect on linear growth of tail feathers or wing was associated with the amount of MeHg in feathers or THg in carcasses. Fledgling tree swallows that survive to migrate, however, will leave Maine with substantial concentrations of Hg in their tissues.