Antennal Responses to Floral Scents in the Butterfly Heliconius melpomene

Floral scent, together with visual floral cues, are important signals to adult butterflies searching for food-rewarding plants. To identify which compounds in a floral scent are more attractive and, thus, of biological importance to foraging butterflies, we applied electrophysiological methods. Antennal responses of male and female adults of the tropical butterfly Heliconius melpomene L. (Lepidoptera: Nymphalidae: Heliconiinae) to individual compounds of natural floral scents and synthetic floral scent mixtures were investigated using gas chromatography–electroantennographic detection (GC-EAD). The natural floral scents included those of two tropical plant species, Lantana camaraL. (Verbenaceae) and Warszewiczia coccinea (Vahl) Kl. (Rubiaceae), and two temperate species, Buddleja davidii Franchet (Loganiaceae) and Cirsium arvense (L.) Scop. (Asteraceae). The two synthetic floral scent mixtures contained many of the compounds found in the natural scents, but all in equal quantities. Compounds both present in relatively high abundance in the floral scents and detected exclusively in the floral parts of the plant, such as linalool, linalool oxide (furanoid) I and II, oxoisophoroneoxide, and phenylacetaldehyde, elicited the strongest antennal responses, suggesting that they may reflect adaptations by the plant to attract butterfly pollinators. However, other compounds also present in high abundance in the floral scent, but detected in the vegetative as well as floral plant parts, either elicited strong antennal responses, such as trans--ocimene and benzaldehyde, or failed to elicit antennal responses, such as the sesquiterpenes -caryophyllene and -humulene from L. camara. The most volatile monoterpene alkenes in the synthetic scent mixtures elicited only low or no responses. Furthermore, the overall antennal responses were stronger in females than in males. The findings suggest that several floral scent volatiles, especially those of exclusively floral origin, are of high biological significance to H. melpomene butterflies. These include compounds of different biosynthetic origins belonging to the benzenoids, monoterpenoids, and irregular terpenoids.     

Variation of Floral Scent Emission and Postpollination Changes in Individual Flowers of Ophrys sphegodes Subsp. sphegodes

We investigated the scent composition of individual flowers of Ophrys sphegodes, its alteration following pollination, and of picked flowers by day and at night. Odor samples were collected by headspace sorption and analyzed by gas chromatography and mass spectrometry. To evaluate the function of postpollination odor changes, we carried out behavioral tests on the pollinator Andrena nigroaenea with pollinated and unpollinated flowers. We identified 27 volatiles in the flower scents. Aldehydes and alkanes were most frequently found. Aldehydes were the most abundant class of compounds (40–50%). When flowers were picked, they emitted significantly lower total amounts of volatiles than unpicked flowers, and their odor bouquets were significantly different. Comparison of scents released by day and at night showed no decrease in scent emission during nighttime, but the odor bouquets were significantly different. Pollinated flowers produced significantly different odor bouquets, and the total amount of scent emitted two to four days after pollination was significantly lower compared with unpollinated flowers. In addition, behavioral tests with A. nigroaenea males showed that flowers were significantly less attractive three days after pollination. This reduced attractiveness is hypothesized to guide pollinators to the unpollinated flowers within an inflorescence, and thus increase the reproductive success of the plant.     

Disease Status and Population Origin Effects on Floral Scent<Emphasis Type="SmallCaps">:</Emphasis> Potential Consequences for Oviposition and Fruit Predation in A Complex Interaction Between A Plant,Fungus, and Noctuid Moth

In the Silene latifolia–Hadena bicruris nursery pollination system, the Hadena moth is both pollinator and seed predator of its host plant. Floral scent, which differs among S. latifolia individuals and populations, is important for adult Hadena to locate its host. However, the success of moth larvae is strongly reduced if hosts are infected by the anther smut fungus Microbotryum violaceum, a pathogen that is transmitted by flower visitors. There were no qualitative differences between the scent of flowers from healthy and diseased plants. In addition, electroantennographic measurements showed that Hadena responded to the same subset of 19 compounds in samples collected from healthy and diseased plants. However, there were significant quantitative differences in scent profiles. Flowers from diseased plants emitted both a lower absolute amount of floral scent and had a different scent pattern, mainly due to their lower absolute amount of lilac aldehyde, whereas their amount of (E)-β-ocimene was similar to that in healthy flowers. Dual choice behavioral wind tunnel tests using differently scented flowers confirmed that moths respond to both qualitative and quantitative aspects of floral scent, suggesting that they could use differences in floral scent between healthy and infected plants to discriminate against diseased plants. Population mean fruit predation rates significantly increased with population mean levels of the emission rates of lilac aldehyde per flower, indicating that selection on floral scent compounds may not only be driven by effects on pollinator attraction but also by effects on fruit predation. However, variation in mean emission rates of scent compounds per flower generally could not explain the higher fruit predation in populations originating from the introduced North American range compared to populations native to Europe.     

Interspecific Variation of Floral Scent Composition in Glochidion and its Association with Host-specific Pollinating Seed Parasite (Epicephala)

Trees of the genus Glochidion (Phyllanthaceae) are pollinated by females of Epicephala moths (Gracillariidae) whose larvae consume the seeds of the flowers that they pollinate. Each Epicephala moth species is specific locally to a single host species, although two to four Glochidion hosts often cooccur. To investigate the role of olfactory signals in maintaining the plant−moth specificity, we analyzed floral scent composition of five Glochidion species by using gas chromatography–mass spectrometry (GC-MS) and conducted Y-tube olfactometer bioassays with Epicephala moths and their host flowers. The GC-MS analysis showed that the floral scents of the five Glochidion species are dominated by (R)-(−)- and (S)-(+)-linalool, and (E)- and (Z)-β-ocimene, and that each species produces 6–20 compounds. Transformation of scent profiles by using chord-normalized expected species shared distances and analysis of the data with nonmetric multidimensional scaling showed that floral volatiles of cooccurring Glochidion species can be distinguished by relative chemical composition, especially that of minor compounds. The bioassay with pollinators of Glochidion lanceolatum and Glochidion ruburm further indicated that Epicephala moths are capable of discriminating their hosts by using floral odor. The results suggest that the floral scent of Glochidion is one of the important key signals that mediate the encounters of the species-specific partners in the Glochidion–Epicephala mutualism.     

Can social bees be influenced to choose a specific feeding station by adding the scent of the station to the hive air?

The behavioral response of honeybees (Apis mellifera L.) and bumblebees (Bombus terrestris L.) to the flower volatiles 2-ethyl-1-hexanol and myrcene isolated in situ from white clover (Trifolium repens L.) and oil seed rape (Brassica napus oleifera), respectively, were investigated on a rotating arena with 12 visually identical, but differently scented, feeding stations. When locating a feeding station, neutral in both shape and color, foragers used scent as orientation cue. Introduction of 2-ethyl-1-hexanol to the honeybee hives induced significantly more visits to sites containing this compound. In contrast, introduction of myrcene to the hives did not influence the foraging choices of honeybees significantly. No effect of hive scent composition on the choices made by bumblebees could be detected. Experienced bumble bees, i.e., bees with more than five visits to the feeding stations, tended to visit a particular position on the arena without discriminating between the two volatiles. In contrast, honeybees showed no positioning behavior on the arena, using primarily odoriferous stimuli. The observed influences of addition of scents to the hives are discussed in relation to the general knowledge on foraging behavior of social bees and the emission of volatiles from leaves and flowers.     

Identification of Floral Volatiles From Ligustrum japonicum that Stimulate Flower-Visiting by Cabbage Butterfly, Pieris rapae

Floral scent compounds of Ligustrum japonicum that affect the foraging behavior of Pieris rapae adults were examined by means of chemical analyses, electroantennogram (EAG) responses, and behavioral bioassays; the behavioral biossays consisted of two tests: reflex extension of proboscis (REP) in response to odor, and attraction to scented and unscented artificial flowers. More than 30 compounds, including 2-phenylethanol, benzyl alcohol, and methyl phenylacetate as the major components were identified from L. japonicum flowers. Of these, 22 compounds were tested for their effect on foraging behavior. Phenylacetaldehyde (PA), 2-phenylethanol (PE), and 6-methylhept-5-en-2-one (MHO) elicited the highest REP responses, and benzaldehyde (BA) and methyl phenylacetate (MPA) evoked intermediate REP responses. EAG responses were not necessarily correlated with REP activities; the three high-REP compounds gave only moderate EAG responses, whereas two other compounds (ethyl phenylacetate and 2-phenylethyl acetate) that released high EAG responses showed low REP activities. In two-choice behavioral bioassays, flower models scented with any one of these high-REP compounds attracted significantly more adults, while compounds with low REP activities exhibited weak or no appreciable attractiveness. This suggests that the REP responsiveness closely reflects the attractiveness of a compound and could be an effective measure in elucidating which chemical attractants are involved in flower-visiting. A synthetic blend of five floral chemicals (PA, PE, MHO, BA, and MPA) displayed an attractiveness that was comparable to that of the floral extract and was more effective in attractiveness than the compounds tested singly. Consequently, it is highly likely that the flower-visiting by P. rapae to L. japonicum is mediated largely by floral scent chemicals and that a synergistic effect of the five floral components would be most responsible for attraction of the butterfly to this flower. The present results also strongly suggest that specific floral volatiles may facilitate close-range flower location by P. rapae, could serve in part as a cue for recognizing food sources, and also be profoundly implicated in flower preference.     

Postpollination Changes in Floral Odor in Silene latifolia: Adaptive Mechanisms for Seed-Predator Avoidance?

Floral odor is a key trait for pollinator attraction in many plants, but may also direct antagonists like herbivores to flowers. In this study, we examined how floral scent changes after pollination in Silene latifolia, which has a specialized relationship with the seed predator Hadena bicruris. We found an overall decrease in total scent emission and considerable changes in relative amounts of scent compounds after pollination. Lilac aldehydes A and B as well as veratrole contributed most to the decrease in scent emission. These three compounds are known to be key signals for the attraction of H. bicruris to the flowers. A specific downregulation of these compounds may increase the reproductive success of the plant by reducing seed predation after pollination.     

Dual role of pyrrolizidine alkaloids in nectar

Pyrrolizidine alkaloids occur in several plant families, attracting ithomiine and danaine butterflies that specialize on the flowers. I show that pyrrolizidine alkaloids in artificial nectar also inhibit at least one butterfly,Agraulis vanillae (Nymphalidae: Heliconiinae), a more general forager. Inhibition was demonstrated in two ways: quantity of nectar consumed and number of artificial flowers visited. The amount of sucrose solution consumed by individual butterflies was measured using a microcapillary tube. Number of flower visits was determined using an array of artificial orange and yellow flowers.A. vanillae drank less sucrose solution with monocrotaline, a pyrrolizidine alkaloid, than without. When monocrotaline was placed into yellow flowers,A. vanillae learned to visit predominantly orange flowers. Evolutionarily, pyrrolizidine alkaloids in nectar may represent an adaptation to exclude butterflies. Ithomiines and danaines, seeking these compounds in larval food plants, were not excluded. Alternatively, ithomiines and danaines overcame the nectar defense. In either case, the plant effectively increased floral constancy by inhibiting generalist butterflies and attracting specialists.     

Flower Scent of Floral Oil-Producing Lysimachia punctata as Attractant for the Oil-Bee Macropis fulvipes

Most flowers offer nectar and/or pollen as a reward for pollinators. However, some plants are known to produce mostly fatty oil in the flowers, instead of nectar. This oil is exclusively collected by specialized oil-bees, the pollinators of the oil-plants. Little is known about chemical communication in this pollination system, especially how the bees find their hosts. We collected the floral and vegetative scent emitted by oil-producing Lysimachia punctata by dynamic headspace, and identified the compounds by gas chromatography coupled to mass spectrometry. Thirty-six compounds were detected in the scent samples, several of which were flower-specific. Pentane extracts of flowers and floral oil were tested on Macropis fulvipes in a biotest. Flower and oil extracts attracted the bees, and some of the compounds identified are seldom found in the floral scent of other plants; these may have been responsible for the attraction of the bees.     

Aqueous Leachate of Lantana camara Kills Water Hyacinth

Lantana camara, a terrestrial plant, was screened for allelopathic impact on water hyacinth (Eichhornia crassipes). Water hyacinth was allowed to grow in experimental pots containing 3% aqueous leachate (w/v) of Lantana twigs. The leachate was allelopathic to the growth of water hyacinth, and killed water hyacinth after 21 days under the experimental conditions. Leachate concentrations from 1–3% of Lantana were highly toxic to water hyacinth plants. Leachate from young Lantana twigs with prickly orange, pink, and yellow flowers (multicolored) was more toxic than leachate from mature twigs. This study indicates the potential for utilization of terrestrial allelopathic species such as Lantana camara to suppress the growth of water hyacinth.     

Identity and Function of Scent Marks Deposited by Foraging Bumblebees

Foraging bumblebees can detect scents left on flowers by previous bumblebee visitors and hence avoid flowers that have been depleted of nectar. Tarsal secretions are probably responsible for this repellent effect. The chemical components of the tarsal glands were analyzed by combined gas chromatography–mass spectrometry for three species of bumblebee, Bombus terrestris, B. lapidarius, and B. pascuorum. The hydrocarbons identified were similar for each species, although there were interspecific differences in the relative amounts of each compound present. The tarsal extracts of all three species comprised complex mixtures of long-chain alkanes and alkenes with between 21 and 29 carbon atoms. When B. terrestris tarsal extracts were applied to flowers and offered to foraging bumblebees of the three species, each exhibited a similar response; concentrated solutions produced a repellent effect, which decreased as the concentration declined. We bioassayed synthetic tricosane (one of the compounds found in the tarsal extracts) at a range of doses to determine whether it gave a similar response. Doses 10^–12 ng/flower resulted in rejection by foraging B. lapidarius. Only when 10^–14 ng was applied did the repellent effect fade. We bioassayed four other synthetic compounds found in tarsal extracts and a mixture of all five compounds to determine which were important in inducing a repellent effect in B. lapidarius workers. All induced repellency but the strength of the response varied; heneicosane was most repellent while tricosene was least repellent. These findings are discussed in relation to previous studies that found that tarsal scent marks were attractive rather than repellent.     

Ability of honeybee,Apis mellifera,to detect and discriminate odors of varieties of canola (Brassica rapa and Brassica napus) and snapdragon flowers (Antirrhinum majus)

Honeybees (Apis mellifera) use odors to identify and discriminate among flowers during foraging. This series of experiments examined the ability of bees to detect and discriminate among the floral odors of different varieties of two species of canola (Brassica rapa and Brassica napus) and also among three varieties of snapdragons (Antirhinnum majus). Individual worker honeybees were trained using a proboscis extension assay. The ability of bees to distinguish a floral odor from an air stimulus during training increased as the number of flowers used during training increased. Bees conditioned to the odor of one variety of flower were asked to discriminate it from the odors of other flowers in two different training assays. Bees were unable to discriminate among flowers at the level of variety in a randomized presentation of a reinforced floral odor and an unreinforced floral odor. In the second type of assay, bees were trained with one floral variety for 40 trials without reinforcement and then tested with the same variety or with other varieties and species. If a bee had been trained with a variety of canola, it was unable to differentiate the odor of one canola flower from the odor of other canola flowers, but it could differentiate canola from the odor of a snapdragon flower. Bees trained with the odor of snapdragon flowers readily differentiated the odor of one variety of a snapdragon from the odor of other varieties of snapdragons and also canola flowers. Our study suggests that both intensity and odor quality affect the ability of honeybees to differentiate among floral perfumes.     

Analysis of the Volatiles Emitted by Whole Flowers and Isolated Flower Organs of the Carob Tree Using HS-SPME-GC/MS

The volatiles emitted by fresh whole flowers and isolated flower organs of male, female, and hermaphrodite carob trees (Ceratonia siliqua L.; Leguminosae) were analyzed by headspace solid-phase microextraction followed by capillary gas chromatography and mass spectrometry. The headspace of carob flowers is mainly constituted of high amounts of monoterpenes and sesquiterpenes, and more than 25 compounds were identified. The gender and cultivar affected both the qualitative profile and the relative abundances of the volatiles of whole flowers and isolated floral organs. Linalool and its derivatives (cis-linalool furan oxide, 2,2,6-trimethyl-3-keto-6-vinyltetrahydropyran, cis-linalool pyran oxide, and trans-linalool furan oxide), α-pinene, and α-farnesene were the dominant volatiles. Female flowers had a higher diversity of volatile compounds than males and hermaphrodites, but a lower abundance of the major ones. Similarly, the floral scent of female flowers of cv. Mulata had a higher content of volatiles but a lower abundance of the major ones, when compared to cv. Galhosa. In each of the three gender types of flowers, the nectary disks seemed to be the major source of volatiles.     

Biosynthetic and Functional Color–Scent Associations in Flowers of Papaver nudicaule and Their Impact on Pollinators

Despite increasing evidence for biosynthetic connections between flower pigments and volatile compounds, examples of such relationships in polymorphic plant species remains limited. Herein, color–scent associations in flowers from Papaver nudicaule (Papaveraceae) have been investigated. The spectral reflectance and scent composition of flowers of four color cultivars was determined. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. Whereas yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. By using the honeybee, Apis mellifera, evaluations were made on how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling those of the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. The results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color–scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators.     

An Antiaphrodisiac in Heliconius melpomene Butterflies

Gilbert (1976) suggested that male-contributed odors of mated females of Heliconius erato could enforce monogamy. We investigated the pheromone system of a relative, Heliconius melpomene, using chemical analysis, behavioral experiments, and feeding experiments with labeled biosynthetic pheromone precursors. The abdominal scent glands of males contained a complex odor bouquet, consisting of the volatile compound (E)-β-ocimene together with some trace components and a less volatile matrix made up predominately of esters of common C₁₆- and C₁₈-fatty acids with the alcohols ethanol, 2-propanol, 1-butanol, isobutanol, 1-hexanol, and (Z)-3-hexenol. This bouquet is formed during the first days after eclosion, and transferred during copulation to the females. Virgin female scent glands do not contain these compounds. The transfer of ocimene and the esters was shown by analysis of butterflies of both sexes before and after copulation. Additional proof was obtained by males fed with labeled D-¹³C₆– glucose. They produced ¹³C-labeled ocimene and transferred it to females during copulation. Behavioral tests with ocimene applied to unmated females showed its repellency to males. The esters did not show such activity, but they moderated the evaporation rate of ocimene. Our investigation showed that β-ocimene is an antiaphrodisiac pheromone of H. melpomene.     

Effects of Volatile Compounds Emitted by Protea Species (Proteaceae) on Antennal Electrophysiological Responses and Attraction of Cetoniine Beetles

Evolutionary shifts in pollination systems within a plant genus are commonly associated with changes in floral scent, reflecting selection mediated through the sensory systems of various pollinators. The most common cetoniine beetle pollinator of grassland Protea species in South Africa, Atrichelaphinis tigrina, previously has been shown to have a strong preference for the fruity floral scent of these plants over the weak scent of their bird-pollinated congeners. However, it is not known which of the many compounds found in the scent of beetle pollinated Protea species play a role for pollinator attraction. Electroantennograms (EAG) from A. tigrina beetles were recorded in response to 15 compounds emitted by Protea flower heads. EAG responses to all 15 compounds were significantly greater than those to the paraffin solvent in which they were diluted. The greatest responses were observed for aromatics (anisole, methyl benzoate, methyl salicylate, benzaldehyde) followed by the monoterpene β-linalool, which can comprise up to 66 % of fruity Protea scents. Five compounds that elicited EAG responses (benzaldehyde, β-linalool, (E/Z)-linalool oxide (furanoid), methyl benzoate, and methyl salicylate) were tested in commercially available yellow bucket traps in the field to test their attractiveness to beetles. Traps baited with methyl benzoate, β-linalool, (E/Z)-linalool oxide (furanoid), and methyl salicylate caught significantly more insects than did those containing paraffin only. Methyl benzoate also was more specifically attractive to A. tigrina than was (E/Z)-linalool oxide (furanoid) and paraffin baited controls. A second field experiment using a combination of linalool vs. paraffin baited yellow or green traps showed that trap color had a significant effect on the number of trapped beetles. Yellow traps yielded a ten-fold higher number of insect catches than did green traps. However, the combination of yellow color and the scent compound linalool yielded the highest number of catches. This study has shown that the cetoniine beetle A. tigrina can detect a variety of floral compounds and is attracted to compounds comprising a large proportion of the blend that makes up fruity Protea scents, adding support for the hypothesis that change in scent chemistry during the shift from bird to cetoniine beetle pollination in this genus were mediated by beetle sensory preferences.     

Attraction ofHeliothis virescens (F.) (Lepidoptera: Noctuidae) to volatiles from extracts of cotton flowers

Mated and virgin female and virgin male tobacco budworm moths,Heliothis virescens (F.), responded via directed upwind flight in wind-tunnel bioassays to volatiles from floral extracts of cotton,Gossypium hirsutum L. Significantly more male and female moths landed on cloth dispensers treated with a methylene chloride extract of debracted flowers than on control dispensers treated only with methylene chloride. Only mated females landed in significant numbers on the dispensers treated with extract of cotton squares, flowers (including bracts), or flower petals. After landing, most of the moths examined the dispensers by probing with their antennae, proboscis, and/or ovipositor.     

Epiphytic Bacteria Alter Floral Scent Emissions

Floral scents are key mediators of biotic interactions between flowers and various organisms such as pollinators, antagonistic animals and bacteria. It has been shown that emissions of floral volatiles are influenced by interactions with other organisms at the levels of roots, leaves and flowers. However, it is largely unknown whether and how epiphytic bacteria associated with flowers affect the composition of floral scent. By comparing volatiles of sterile and inoculated plants we found that bacteria may add components, induce or reduce the emission of compounds, and potentially catabolize others. These mechanisms collectively altered the floral scent emission and led to clearly different compositions. Our results confirm that bacteria have the potential to interfere with flower-animal interactions with consequences for pollination and plant reproduction.     

Dihydropyrrolizine attractants for arctiid moths that visit plants containing pyrrolizidine alkaloids

Adults of three species of arctiid moths (Cisseps fulvicollis, Ctenucha virginia, andHalysidota tessellaris) are attracted to plants that contain pyrrolizidine alkaloids (PAs). The moths use olfactory cues to locate these plants, then feed on leaves, flowers, and roots with the proboscis. To investigate the chemical basis of attraction, sticky traps were baited with roots of a PA-containing plant,Eupatorium maculatum, alkaloids ofE. maculatum, and several derivatives of these alkaloids. Volatile derivatives of the bicyclic pyrrolizidine skeleton attracted all three arctiid species. The dihydropyrrolizines, (S)-(+)-hydroxydanaidal and (R)-(–)-hydroxydanaidal, proved to be the most attractive compounds tested, accounting for over 70% of the moths captured. Different alkaloid derivatives attracted different proportions of male and femaleCisseps. Both (S)-(+)-hydroxydanaidal (52% male) and (R)-(–)-hydroxydanaidal (71% male) attracted a significantly lower percentage ofCisseps males thanE. maculatum roots (87% male).Cisseps males possess eversible scent organs (coremata) that are displayed during courtship. Analysis of corematal extracts revealed the presence of hydroxydanaidal.Cisseps moths thus resemble danaine and ithomiine butterflies, both in their attraction to PA sources and in the presence of PA derivatives in the male scent organs.     

Field Attraction of Hoplia communis to 2-Phenylethanol, A Major Volatile Component from Host Flowers, Rosa spp.

The attractiveness of volatile compounds from the floral scent of Rosa, one of the most preferred plants for adult Hoplia communis, was evaluated under field conditions. The beetles were attracted to most compounds tested, but 2-phenylethanol exhibited the highest capture rate. Catches increased with increasing emissions of between 9.1 and 287.2 mg/day. Catches in white traps were significantly larger (17.4-fold) than those in green traps when both were baited with anethole, an already known attractant; however, the trap color was not significant when a more attractive lure, 2-phenylethanol, was used. The use of a single funnel trap baited with 2 g of 2-phenylethanol at a heavily infested nursery exhibited promising results for mass trapping. Approximately 90,000 beetles of both sexes, which nearly corresponds to the estimated maximum population per 1000 m², were captured within six days.