Tracing Pollinator Footprints on Natural Flowers

Many insects are known to leave lipid footprints while walking on smooth surfaces. Presumably, the deposited substances improve tarsal adhesion. In bumblebees, footprint hydrocarbons also function as scent marks that allow detection and avoidance of recently depleted flowers. I used GC-MS to detect hydrocarbons deposited by bumblebee (Bombus pascuorum) on flowers of Lamium maculatum. In addition to the plants' own cuticular lipids, extracts of corollas that had been visited by bumblebees contained odd-numbered alkenes. The amount of pentacosenes (C₂₅H₅₀) on corollas was linearly related to the number of bumblebee visits, with workers depositing approximately 16 ng per visit (extrapolated to a total of 65 ng of bumblebee cuticular hydrocarbons). Pentacosenes were retained on visited flowers without loss for 2 hr, and probably longer. This and results from flight cage experiments suggest that flower epicuticles retain a chemical record of pollinator visitation, including information on visiting bee species. Continuous footprint accumulation necessitates new explanations concerning the reversibility of “repellent scent marks” of bumblebees.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

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.     

Host recognition and the study of a chemical basis for attraction by cuckoo bumble bees (Hymenoptera: Apidae)

Species ofPsithyrus (Hymenoptera; Apidae) are obligate bumble bee social parasites. In this study, females ofP. vestalis andP. ashtoni were presented with pentane extracts prepared from different body parts of queens of their respective host species,Bombus terrestris andB. terricola. Parasites of both species were capable of distinguishing host bees from other bumble bee species using chemical cues contained within extracts. Among extracts of several body parts presented to parasites, the abdomen produced the greatest behavioral response, with Dufour's gland and terminal tergal segments eliciting the greatest response among abdominal regions. Extracts of these two body parts obtained fromB. terrestris queens shared a number of compounds, identified by GC-MS. Among the identified compounds are a number that have been reported to be of importance in bee sociochemistry.     

Identification of Female Sex Pheromone of the Rice Leaf Bug, Trigonotylus caelestialium

The female sex pheromone of the rice leaf bug, Trigonotylus caelestialium, was analyzed by GC-EAD and GC-MS. Ten EAD active compounds—n-hexyl n-hexanoate, (E)-2-hexenyl n-hexanoate, n-hexyl (E)-2-hexenoate, n-octyl n-butyrate, n-octyl n-hexanoate, (E)-2-octenyl n-hexanoate, n-pentyl n-hexanoate, n-hexyl n-butyrate, (E)-2-hexenyl n-butyrate, and n-hexyl (E)-2-butenoate—were detected in the ratio of 1000:414–491:trace–5:5–11:55–71:50–63:trace–3:225:90:32 from female body extracts, and in the ratio of 1000:271–342:10–43:1–3:58–78:14–19:trace:178:36:26 from male body extracts. Field trapping tests with these synthetic compounds indicated that n-hexyl n-hexanoate, (E)-2-hexenyl n-hexanoate, and n-octyl n-butyrate are pheromone components, and mixtures in ratios of 1000:400–500:10–100 were more attractive to males. Doses ranging from 4.29 g to 14.3 g of the three-component mixture in the ratio of 1000:400:30 loaded into glass capillary tubes were most attractive to males.     

Squash leaf glandular trichome volatiles: Identification and influence on behavior of female pickleworm moth [Diaphania nitidalis (Stoll.)] (Lepidoptera: Pyralidae)

Fourteen volatile compounds occurring in leaf trichomes of yellow squash (Cucurbita pepo L. cv. Early Prolific Straightneck) were identified. These compounds accounted for 83.5% of the volatile matrix. Ubiquitous constituents of the epidermis (myristic, palmitic, and stearic acids,n-tricosane, andn-pentacosane) accounted for 73.7%; these compounds were not bioassayed. The volatileso-,m-, andp-xylene, toluene, 2-heptanone, (R)-(+)- and (S)-(–)-limonene, and germacrene D were tested for their influence on attraction and oviposition by the pickleworm moth (Diaphania nitidalis Stoll.). No single compound, except germacrene D, was attractive. (R)-(+)-Limonene and 2-heptanone were weakly repellent. Mixtures of the highly volatile fractions were as attractive as volatiles emanating from whole, intact leaves. Oviposition levels on treated artificial sites corresponded with levels of visitation. Oviposition was significantly stimulated by whole-leaf volatiles, and (S)-(–)-limonene caused a slight but significant reduction.Mention of a trade name or proprietary product does not constitute a warranty or guarantee by the U.S. Department of Agriculture, nor does it imply exclusion of other products that may also be suitable.     

Wax Lipids Signal Nest Identity in Bumblebee Colonies

The signalling functions of cuticular lipids, particularly cuticular hydrocarbons, have gained considerable attention in social insect communication. Information transfer between individuals by means of these substances has been examined extensively. However, communication with cuticular lipids is not limited to inter-individual recognition. Cuticular compounds can also have a signalling function in the nest environment. Workers of the bumblebee Bombus terrestris leave cuticular lipid traces, so-called footprints, that mark their nest entrance. In addition, there is evidence that bumblebees sense nesting material to identify their colony. In this study, we examined the signalling potential of bumblebee wax, and tested if bumblebee workers are able to identify their colony with the help of wax scent. Chemical analyses of wax extracts using coupled gas chromatography–mass spectrometry showed that wax from colonies of the bumblebee B. terrestris contained a complex blend of cuticular lipids, dominated by hydrocarbons and wax esters. Comparing the relative compound amounts of wax samples from different colonies, we found that wax scent patterns varied with nest identity. Olfactometer bioassays showed that bumblebees were able to discriminate between wax scents from their own and a foreign colony. Our findings suggest that wax emits characteristic olfactory profiles that are used by workers to recognize their colony.     

Semiochemicals from bark beetles: New results,remarks, and reflections

A brief survey is given about recent results in the identification of semiochemicals in bark beetles: Males ofIps sexdentatus (Boern.), stressed by the attack on resinous trees produce large amounts of 3(S)-1-methyl-5-(1-hydroxyl-1-methylethyl)-cyclohexa-1,3-diene. The compound appears to be derived from ³-carene and acts as a repellent. Males ofIps typographus (L.), stressed through the attack on unsuitable host material release 3-methyl-7-methylene-1,3(E), 8-nonatriene, which seems to act as a repellent. The odor bouquet of three species ofPityogenes is described. The occurrence of (+)-grandisol and other compounds related to weevil pheromones points to a close relation between Scolytidae and Curculionidae. Females ofDendroctonus simplex (Le Conte) use (–)-frontalin as the main pheromone. 6-Methyl-6-hepten-2-one, a minor component among the volatile compounds released by the females, is regarded as a possible precursor of frontalin. Similarly, (2R,5S)-2(1-hydroxyl-1-methylethyl)-5-methyltetrahydrofuran, pityol, a pheromone ofPityophthorus spp., is regarded to at least share a common biogenetic precursor with 6-methyl-5-hepten-2-ol, sulcatol. A new bicylic acetal, 2-ethyl-1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane, is described as an aggregation pheromone of the beech bark beetle,Taphrorychus bicolor (Herbst). Structural relationships between bark beetle pheromones and plant volatiles are discussed.     

Isolation and identification of house fly,Musca domestica L., repellents from pepper tree,Schinus molle L.

Foliage from the pepper tree,Schinus molle L., is traditionally used in Ethiopia to repel house flies,Musca domestica L. The volatile extracts of pepper tree leaves were shown to have repellent and feeding-deterrent activity against house flies in a two-choice laboratory bioassay. High-performance liquid chromatographic fractionation of steam-distilled volatiles from leaves, monitored by laboratory bioassays, demonstrated that bioactivity is associated with two compounds,cis-menth-2-en-1-ol andtrans-piperitol. The absolute configuration of the latter was established as (1S,6S)-piperitol by comparison of acetyl lactate derivatives. Racemic compounds were synthesized from piperitone, and bioassays with house flies indicatedtrans-piperitol to be the most active house fly repellent.     

Chemical Ecology of the Defense of Two Nymphalid Butterfly Larvae Against Ants

We analyzed the behavioral responses of the ants Camponotus rufipes and Solenopsis geminata towards all instars of Dione junio and Abananote hylonome. We also analyzed ant behavior towards hexane extracts of larvae and extracts of the spines and neck glands of the fifth instars of both species and identified the chemical compounds present. Larvae of both species were repellent to ants from the first instar onward. Later instars survived ant attacks better than earlier instars. The spines and neck glands of the larvae influenced the behavior of C. rufipes. The chemical compounds contained in the hexane extracts of whole first and fifth instars and in the spines and neck glands of fifth instars were principally carboxylic acids and terpenes. Further bioassays confirmed the repellent effect of some of these acids toward ants.     

Electroantennogram responses ofHyles lineata (Sphingidae: Lepidoptera) to volatile compounds fromClarkia breweri (Onagraceae) and other moth-pollinated flowers

Electroantennograms (EAGs) from field-collectedHyles lineata moths were recorded in response to 10 individual floral volatiles identified fromClarkia breweri (Onagraceae), to 22 scent compounds produced by other moth-pollinated flowers and to eight ubiquitous green leaf volatiles. Females' EAGs were generally 1.5- to 2-fold greater than those observed for male moths. Female:male EAG rank orders were significantly correlated, but marked differences in order were observed for some compounds (e.g., benzyl alcohol, cinnamic aldehyde, geraniol, and linalool). Linalool, benzyl acetate, methyl salicylate, and pyranoid linalool oxide elicited the largest EAG responses (–1.2 to –0.8 mV) among scent compounds fromC. breweri. EAG responses were significantly lower for monoterpenes as a pooled compound class than for aromatic esters, alcohols and aldehydes, fatty acid derivatives, N-bearing compounds and oxygenated terpenoids. EAG responses to structurally related scent compounds were not significantly different in most cases. Both male and femaleH. lineata were sensitive to mostC. breweri scent compounds at 10^–2 to 10^–4 µg/µl doses, and rank order in potency varied with the dose/concentration tested.H. lineata's olfactory sensitivity to diverse volatile compounds across a range of doses/concentrations suggests that a broad array of volatiles could function as floral attractants for foraging hawkmoths.     

Scent Marking in Male Premating Behavior of Bombus confusus

Premating behavior in the species Bombus confusus Schenck, 1859 was studied. The marking habits of bumblebee males were revealed and the use of their labial gland secretion in scent marks was proved chemically. Identical compounds were present both in the labial gland secretion and on male-marked objects. This chemical proof is reported for the first time from natural conditions of a B. confusus locality. Males' flight activities, site tenacity, and male–male interactions are described in detail. Contrary to literature reports, male B. confusus behavior does not basically differ from that of already known perching bumblebee species.     

Isolation and identification of (3-methoxyphenyl)acetonitrile as a phytotoxin from meadowfoam (Limnanthes alba) seedmeal

Ethyl ether, ethanol, and water extracts of meadowfoam (Limnanthes alba Hartweg ex. Benth.) seedmeal were prepared and bioassayed against velvetleaf (Abutilon theophrasti Medicus) and wheat (Triticum aestivum L. Cardinal). Both the ethyl ether and ethanol fractions, but not the water extract, inhibited velvetleaf and wheat radicle elongation. Fractionation of the extracts indicated that (3-methoxyphenyl)acetonitrile (3-MPAN) was the active compound from both extracts, comprising >97% of the active ethanol fraction. 3-Methoxybenzyl isothiocyanate, which had been previously shown to be the major breakdown product of glucolimnanthin, the majorL. alba glucosinolate, was not detected in either extract. Radicle elongation of velvetleaf and wheat were inhibited by 3-MPAN with I₅₀ (the concentration required to inhibit growth by 50%) values of approximately 4 × 10^–4 M (velvetleaf) and 7×10^–4 M (wheat).Mention of firms or products does not imply endorsement by the U.S. Department of Agriculture over other firms or products not mentioned.     

Allelochemical regulation of reproduction and seed germination of two BrazilianBaccharis species by phytotoxic trichothecenes

The potent phytotoxic trichothecene roridins and baccharinoids occur naturally in the Brazilian plants,Baccharis coridifolia andB. megapotamica. Biosynthesis of roridins inB. coridifolia appears to be linked to pollination, and the phytotoxins then accumulate in the seed. The roles of the phytotoxins in pollination, seed maturation, and germination of theBaccharis species were investigated. The high production of roridins occurred only in seeds resulting from intraspecific pollination, and the concentration of the toxins in the seeds generally increased with seed maturity. Removal of seed coats from trichothecene-producing BrazilianBaccharis species (B. coridifolia andB. megapotamica) and non-trichothecene-producing AmericanBaccharis species (B. halimifolia andB. glutinosa) resulted in improved seed germination ofB. halimifolia andB. glutinosa but complete inhibition of seed germination ofB. coridifolia andB. megapotamica. Addition of seed coat extracts of the BrazilianBaccharis species of dilute solutions (10^–6g/ml) of roridins or baccharinoids to the decoated seeds ofB. coridifolia andB. megapotamica resulted in germination, while seeds ofB. halimifolia andB. glutinosa were killed by the phytotoxins. Roridins interacted with gibberellic acid, a germination promoter, but not with abscisic acid, a germination inhibitor. The results from this study suggest that macrocyclic trichothecenes have a regulatory role(s) on reproduction and germination of BrazilianBaccharis species in their natural habitat.     

An evaluation of repellents to suppress browsing by possums

The efficacy in feeding suppression trials with possums (Trichosorus vulpecula Kerr) of six synthetic odorous chemicals uniquely associated with various of the Mustelidae species and of the red fox (Vulpes vulpes) has been evaluated with respect to several proprietary repellents. The predator odors, two proprietary repellents, and two other repellent formulations (olive oil in petroleum jelly and a synthetic fermented egg odor cocktail) were applied as 5% w/v solutions in paraffin to the foliage ofPinus radiata seedlings and the extent of browse compared with that on seedlings treated with Treepel, an effective egg-based repellent already available commercially. All four of the mustelid predator odors [()-2-n-propylthietane, ()-3-n-propyl-1,2-dithiolane, 2,2-dimethylthietane, and 3,3-dimethyl-1,2-dithiolane] and the volatile compound from fox urine (³-isopentenyl methyl sulfide) were shown to be more or at least equally effective compared to Treepel. This was true also of one commercial repellent Gori, which contained bitrex and the synthetic fermented egg formulation. The most effective compounds will be evaluated when administered with adhesives and surfactants in the further development of possum repellents with longer field lives.     

Induction and Systemic Release of Herbivore-Induced Plant Volatiles Mediating In-Flight Orientation of Aphidius ervi

In-flight orientation of the braconid Aphidius ervi in response to volatiles released from broad bean plants infested by the pea aphid, Acyrthosiphon pisum, was studied in a no-choice wind-tunnel bioassay. The role of aphid infestation level and duration, systemic production of volatiles by insect-free parts of the plant, and the specificity of aphid-induced volatiles on the flight behavior of the foraging female parasitoids were investigated. The upper insect-free part of a three-leaved broad bean plant, which was basally infested by a population of 40 A. pisum, released synomones detectable by A. ervi females after at least 48–72 hr of infestation, resulting in both significant increases in oriented flights and landings on the source compared with uninfested control plants. This suggests that volatiles involved in host-location by A. ervi are systemically released by broad bean plants either in response to circulation of aphid saliva, circulation of saliva-induced bioactive elicitors, or circulation of the synomones themselves. Air entrainment extracts of volatiles collected from a broad bean plant infested by the nonhost Aphis fabae or an uninfested broad bean plant elicited few oriented flights and landing responses by female parasitoids. These extracts were significantly less attractive than extracts collected from a broad bean plant infested by the host A. pisum, indicating the specificity of synomones elicited by different aphid species on the same plant species.     

Cereal leaf beetle response to biochemicals from barley and pea seedlings. I. Crude extract,hydrophobic and hydrophilic fractions

To identify contact feeding stimulants for adult cereal leaf beetles (CLB),Oulema melanopus (L.), compounds extracted from a host plant,Hordeum vulgare L. (cultivar Lakeland), were bioassayed in 3% agar. Damage to agar gel with the hydrophilic fraction was dominated by narrow channels; the hydrophobic fraction gel was dominated by bites, rashes, and shorter channels. This difference probably reflected the functional nature of compounds in each fraction. Crude extract and the hydrophobic fraction of nonhost pea seedlings,Pisum sativum L., prevented CLB feeding behavior, but the hydrophilic fraction only reduced it. Combination of barley and pea crude extracts, by weight at 11 or greater, evoked a small feeding response which indicated a host-specific recognition factor in the barley.Michigan Agricultural Experiment Station Journal Article No. 8227.The use of a trade name in this paper does nit constitute an official endorsement or approval by the USDA.     

Alarm pheromones from the marine opisthobranchNavanax inermis

When molested, the marine opisthobranchNavanax inermis secretes into its slime trail, a bright yellow mixture of three major compounds and several closely related minor substances. Collectively, these compounds induce an avoidance-alarm response in a trail-followingNavanax at the concentration limits of 1 × 10^–5 M. The three major compounds have been isolated and identified as 10-(3-pyridyl)-3E,5E,7E,9E-decatetraen-2-one (navenone A), 10-phenyl-3E,5E,7E,9E-decatetraen-2-one (navenone B) and 10-(4-hydroxyphenyl)-3E,5E,7E,9E-decatetraen-2-one (navenone C). The minor constituents of the mixture are proposed as the 3Z,5Z,7E,9E-isomers and the 3-methyl homologs of navenones A and B. The navenones appear to be produced in a specialized gland referred to in earlier studies as the yellow gland and to communicate the presence of predators within the species.     

Floral Scent of Osmanthus fragrans Discourages Foraging Behavior of Cabbage Butterfly, Pieris rapae

Field observations revealed that flowers of Osmanthus fragrans attract few insect species despite their strong scent and vivid coloration (yellowish orange). Floral volatiles of the plant were thus examined for suspected repellency to Pieris rapae, a potential visitor that never visits this flower. The influence of odor components on the foraging behavior of adult butterflies was assessed by two behavioral bioassays: proboscis extension reflex (PER) in response to olfactory stimuli and the frequency of visits to artificial flowers. In the PER bioassays, a binary mixture composed of a test material and 2-phenylethanol (a positive standard compound) was used, and the negative effect of test materials was appraised based on the degree of suppression caused compared with the original PER performance evoked by 2-phenylethanol alone. Isopentane extract of the flower exhibited significant deterrency in the PER test and remarkable repellency in the flower-visiting experiment, thereby suggesting the presence of a repellent(s) in the floral volatiles. Fractionation of the extract by silica gel column chromatography yielded an active fraction (based on PER performance), which, upon GC and GC-MS analyses, was found to consist of -decalactone, -ionone, and linalool oxide (LO) isomers (furanosides and pyranosides) as major components together with small amounts of linalool and -ionone. -Decalactone was strongly deterrent in the PER test, and -ionone and all LO isomers were weakly deterrent, while the other compounds exhibited no deterrency. In the flower-visiting tests, -decalactone showed notable repellency, whereas -ionone had no repellent effect. Among the LO isomers, at least furanosides (cis/trans mixture) and cis-pyranoside were found to be weakly repellent. On the other hand, the butterfly showed strong to weak EAG responses to LO furanosides, cis-LO pyranoside, -decalactone, and -ionone in decreasing order of intensity. The present results clearly indicate that the repellency of a compound is correlated with its deterrent effect on PER but not necessarily with antennal sensitivity.