Post-pollination emission of a repellent compound in a sexually deceptive orchid: a new mechanism for maximising reproductive success?

The flowers of the sexually deceptive orchid Ophrys sphegodes are pollinated by pseudocopulating males of the solitary bee Andrena nigroaenea. We investigated the changes in odor emission and reduced attractiveness that occur after pollination in these plants. We analyzed floral odor of unpollinated and pollinated flowers by gas chromatography and compared relative and absolute amounts of electrophysiologically active compounds. Headspace odor samples of O. sphegodes flowers showed a significant increase in absolute and relative amounts of all-trans-farnesyl hexanoate after pollination. Flower extracts also indicated an increase of farnesyl hexanoate after pollination. The total amount of the other physiologically active odor compounds decreased slightly. Farnesyl hexanoate is a major constituent of the Dufour's gland secretion in females of the pollinator bees, A. nigroaenea, where it functions in the lining of the brood cells. Furthermore, this compound lowers the number of copulation attempts in males. In dual-choice tests, we showed that flowers artificially scented with an amount of farnesyl hexanoate equal to the increased amount after pollination were significantly less attractive than flowers treated with solvent only. We propose that the increased production of farnesyl hexanoate in pollinated flowers is a signal to guide pollinators to unpollinated flowers of the inflorescence, which represents a new mechanism in this pollination system.     

Pollinator attracting odour signals in sexually deceptive orchids of the Ophrys fusca group

We investigated patterns of volatiles of several allopatric and sympatric species of the Ophrys fusca group and one species of the O. mammosa/sphegodes group pollinated by either Andrena nigroaenea or A. flavipes, using electrophysiology (gas chromatography coupled with electroantennography; GC-EAD) and chemical analyses. We found 52 GC-EAD active compounds, mainly saturated and unsaturated hydrocarbons with chain lengths of 21 to 31, aldehydes, an ester, and an acid. Based on the relative proportions of all GC-EAD active compounds, the investigated species were compared using various statistical methods (ANOVA, principle component analyses, discriminant function analyses and cluster analyses). Our results show that Ophrys species with the same pollinator – independent of their phylogenetic relationship–use the same volatiles for pollinator attraction. Differences between the species mainly involve different quantitative patterns of volatiles. Our results are in congruence with previous studies that showed different odour bouquets to be responsible for the specific attraction of different pollinators and that alkanes and alkenes are most important for pollinator attraction.     

Do changes in floral odor cause speciation in sexually deceptive orchids?

 We investigated differences in floral odor between two sympatric, closely related sexually deceptive orchid species, Ophrys fusca and O. bilunulata, which are specifically pollinated by Andrena nigroaenea and A. flavipes, respectively. We identified biologically active compounds by gas chromatography with electroantennographic detection using antennae of the pollinator bees. Alkanes, alkenes, aldehydes, and farnesyl hexanoate released electroantennographic reactions. The relative amounts of alkanes were mostly the same between the two orchid species, whereas the relative amounts of most alkenes were significantly different. On the grounds of these findings and behavioral experiments conducted in earlier studies, we suggest that the difference in relative amounts of alkenes is responsible for the selective attraction of pollinators in the two orchids. Speciation in this group of Ophrys orchids may be brought about by changes in pattern of alkenes, which lead to attraction of a different pollinator species and therefore reproductive isolation. Received November 22, 2001; accepted February 21, 2002 Published online: November 7, 2002 Addresses of the authors: Florian P. Schiestl* and Manfred Ayasse, Department of Evolutionary Biology, Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna. *Present address: Geobotanical Institute ETH, Zollikerstrasse 107, CH-8008 Zürich. (e-mail: schiestl@geobot.umnw.ethz.ch)     

Pollinator shifts between Ophrys sphegodes populations: might adaptation to different pollinators drive population divergence?

Local adaptation to different pollinators is considered one of the possible initial stages of ecological speciation as reproductive isolation is a by‐product of the divergence in pollination systems. However, pollinator‐mediated divergent selection will not necessarily result in complete reproductive isolation, because incipient speciation is often overcome by gene flow. We investigated the potential of pollinator shift in the sexually deceptive orchids Ophrys sphegodes and Ophrys exaltata and compared the levels of floral isolation vs. genetic distance among populations with contrasting predominant pollinators. We analysed floral hydrocarbons as a proxy for floral divergence between populations. Floral adoption of pollinators and their fidelity was tested using pollinator choice experiments. Interpopulation gene flow and population differentiation levels were estimated using AFLP markers. The Tyrrhenian O. sphegodes population preferentially attracted the pollinator bee Andrena bimaculata, whereas the Adriatic O. sphegodes population exclusively attracted A. nigroaenea. Significant differences in scent component proportions were identified in O. sphegodes populations that attracted different preferred pollinators. High interpopulation gene flow was detected, but populations were genetically structured at species level. The high interpopulation gene flow levels independent of preferred pollinators suggest that local adaptation to different pollinators has not (yet) generated detectable genome‐wide separation. Alternatively, despite extensive gene flow, few genes underlying floral isolation remain differentiated as a consequence of divergent selection. Different pollination ecotypes in O. sphegodes might represent a local selective response imposed by temporal variation in a geographical mosaic of pollinators as a consequence of the frequent disturbance regimes typical of Ophrys habitats.     

A blend of formic acid,benzoic acid,and aliphatic alkanes mediates alarm recruitment responses in western carpenter ants,Camponotus modoc

Formicine ants in distress spray alarm pheromone which typically recruits nestmates for help. Studying the western carpenter ant, Camponotus modoc Wheeler (Hymenoptera: Formicidae), our objectives were to (1) determine the exocrine glands that contain alarm recruitment pheromone, (2) identify the key alarm recruitment pheromone components, and (3) ascertain the pheromone components that are discharged by distressed ants. In Y-tube olfactometer experiments, extracts of poison glands, but not of Dufour’s glands, elicited anemotactic responses from worker ants. Gas chromatographic-mass spectrometric analyses of poison gland extracts revealed the presence of (1) aliphatic alkanes (undecane, tridecane, pentadecane, heptadecane), (2) aliphatic alkenes [(Z)-7-pentadecene, (Z)-7- and (Z)-8-heptadecene], (3) two acids (formic, benzoic), and (4) other oxygenated compounds (hexadecan-1-ol, hexadecyl formate, hexadecyl acetate). Testing the responses of worker ants in Y-tube olfactometers to complete and partial synthetic blends of these compounds revealed that the acids and the alkanes are essential alarm pheromone components. In two-choice arena bioassays, micro-locations treated with synthetic alarm pheromone recruited worker ants. Acids and alkanes were abundant in the poison gland and the Dufour’s gland, respectively, suggesting that the alarm pheromone components originate from both glands. Moreover, alarm pheromone sprays of ants differed in that all sprays contained formic acid but only some also contained alkanes, implying that ants can independently discharge the content of either one or both glands in accordance with the type of distress incident they experience.     

Pollination ofOphrys (Orchidaceae) in Cyprus

In the southern part of Cyprus the pollinator —Ophrys (Orchidaceae) relationships and its specifity have been investigated from the end of February until the middle of March 1986. 12Ophrys spp. were found. To date, only a single pollinator reference has been reported from this island. We found the following pollinators:Melecta tuberculata (Ophrys kotschyi),Eucera dimidiata (Ophrys flavomarginata),Eucera gaullei (Ophrys umbilicata),Eucera paulusi (Ophrys bornmuelleri),Anthophora erschowi (Ophrys elegans),Andrena torda (Ophrys sicula =O. lutea subsp.minor),Andrena cinereophila (Ophrys fusca, small-flowered),Andrena flavipes (Ophrys israelitica),Andrena morio (Ophrys iricolor andOphrys transhyrcana),Andrena bimaculata (Ophrys sphegodes aggr., probably formerly confused withO. transhyrcana). Most interestingly, it could be verified thatO. flavomarginata/O. umbilicata, O. bornmuelleri/O. levantina andO. transhyrcana/O. sphegodes aggr. (possiblyO. sintenisii) are different biospecies. This is a result of genetic isolation due to varying pollinators, and of differences in flower morphology.     

Induction of balling in worker honeybees (Apis mellifera L.) by “stress” pheromone from Koschewnikow glands of queen bees: Behavioural,structural and chemical study

• 1.1. The Koschewnikow glands (KG) of honeybee queens are composed of type III glandular units; secretions are emitted from ducts that open onto the entire surface of the intersegmental membrane.
• 2.2. Secretory activity is characterized by the appearance of dense granules containing mainly glycoproteins, stained by periodic acid-thiocarbohydrazide-silver proteinate, extracted by pronase. In one-yearold mated queens, the gland degenerates. Secretory products originating from mitochondria are likely to be only carriers of pheromonal compounds.
• 3.3. Topical treatment of worker bees (“pseudoqueens”) with EtOH extracts of queen Koschewnikow glands induced typical queen balling behaviour in workers of a bee colony.
• 4.4. Twenty-eight compounds including acids, alcohols, alkanes and alkenes (C₈H₁₆C₄₃H₈₈) were characterized by gas liquid chromatography-mass spectrometry in queen KG extract. None of them is present in worker alarm pheromone which is secreted from worker KG.

     

Electroantennogram and behavioral responses of workers of the stingless bee Oxytrigona mediorufa to mandibular gland volatiles

Gas chromatography-mass spectrometric analysis showed that the mandibular gland secretion of workers of the stingless bee Oxytrigona mediorufa (Cockerell) (Hymenoptera: Apidae) is composed of 3-hepten-2,5-dione, 3-nonene-2,5-dione, 2-heptanone, hydrocarbons, tetradecyl, and hexadecyl acetates and minor compounds related to these. Behavioral and electroantennographic tests were carried out on conspecific workers to observe the effect of worker mandibular gland secretion, individual compounds (2-heptanone, 2-nonanone, and tetradecyl acetate), and a blend of these three. Bioassays showed that biting responses of O. mediorufa to the odor of the mandibular gland secretion were similar to that of 2-heptanone and to the three-component blend. Oxytrigona mediorufa workers exhibited antennal olfactory responses to the gland extract, 2-heptanone, and the synthetic mixture. The function of the mandibular gland secretion and 2-heptanone is discussed in terms of an alarm pheromone and defensive substances.     

Sex pheromone of the red clover casebearer moth,Coleophora deauratella,an invasive pest of clover in Canada

The red clover casebearer, Coleophora deauratella Lienig & Zeller (Lepidoptera: Coleophoridae), is an invasive pest of Trifolium species (Fabaceae) in Canada. We identified candidate sex pheromone components from female pheromone gland extracts using coupled gas chromatographic–electroantennographic analysis detection. Three compounds elicited an electrophysiological response from antennae and were identified as: (Z)‐7‐dodecenyl acetate, (Z)‐5‐dodecenyl acetate, and (Z)‐7‐dodecen‐1‐ol. Field tests of the candidate pheromone components revealed that males were attracted to a binary mixture of (Z)‐7‐dodecenyl acetate and (Z)‐5‐dodecenyl acetate. Male moth trap capture was greatest in traps baited with lures containing 100:10 or 100:20 ratios of these pheromone components, respectively. Trap capture was reduced when (Z)‐5‐dodecenyl acetate was present below 10 or above 20% of (Z)‐7‐dodecenyl acetate. Equal numbers of male moths were captured in traps baited with 10, 100, and 1 000 μg of the attractive binary mixture. These findings allow for the development of a pheromone‐based monitoring system for this invasive pest of clover in Canada.     

Seasonal stability and species specificity of environmentally acquired chemical mating signals in orchid bees

Traits that mediate reproductive isolation between species, such as those involved in mate choice and/or recognition, are predicted to experience stabilizing selection towards the species mean. Male orchid bees collect chemical compounds from many sources, such as plants and fungi, which they use as a perfume signal (pheromone) during courtship display, and are suggested to contribute to reproductive isolation between species. Environmentally acquired signals are more prone to variation as source availability can vary through space and time. If orchid bee perfumes are important for reproductive isolation between species, we expect them to exhibit stable species-specific differences in time and space. Here, we describe phenotypic patterns of inter- and intraspecific variation in the male perfumes of three sympatric species of Euglossa orchid bees across an entire year, investigating both their seasonality and species specificity. Our analysis revealed considerable within-species variation in perfumes. However, species specificity was maintained consistently throughout the year, supporting the idea that these perfumes could play an important role in reproductive isolation and are experiencing stabilizing selection towards a species mean. Our analysis also identified strong correlations in the abundance of some compounds, possibly due to shared collection sources between species. Our study suggests that orchid bee perfumes are robust in the face of environmental changes in resource availability and thus can maintain reproductive isolation between species.     

Relationships between wild bee abundance and fruit set of Camellia oleifera Abel

Camellia oleifera Abel. is an economically important plant for edible oils. To investigate the importance of wild bee abundance at different canopy and spatial scale for fruit set of Camellia oleifera, we evaluated the relationship of bee abundance with fruit set using yellow pan trap, with additional focus on distance and wild bee abundance counts at a farm in China for 2 years. We found that yellow traps collected 95% more individual bees than the second-best (pink) trap colour. Looking across canopy positions, the number of wild bees (including Colletes gigas, Andrena camellia, Andrena striata, Andrena hunanensis and Andrena chekiangensis) varied significantly in each year. The fruit set also varied significantly based on canopy position. In 2018, there was with an average fruit set of 34.13% in the lower canopy, 54.30% in the middle canopy and 47.41% in the upper canopy. In 2019, fruit set was 29.67% in the lower canopy, 54.15% in the middle canopy and 47.36% in the upper canopy. Comparing inter-annual changes, only the fruit set and number of bees in the low canopy showed significant differences between 2018 and 2019. Fruit set and wild bee abundance were positively correlated in all canopy layers in each year, and there was an interactive effect between canopy and bee abundance on fruit set. Across 205 trees, growth indicators, crown size and tree height, increased significantly from 2018 to 2019, but the fruit set did not increase with the significant increase in the number of flowers. From the spatial perspective, wild bee abundance and fruit set were positively correlated, but fruit set was negatively correlated with distance from a large bee nesting aggregation nearby. Among the variables investigated, bee abundance seems to have the strongest influence on fruit set in C. oleifera.     

Floral evolution and pollinator mate choice in a sexually deceptive orchid

Sexually deceptive orchids mimic sex pheromones and appearance of female insects to attract males, which pollinate the flowers in an attempted mating. This study examines the effects of pollinator mate choice on orchid floral evolution using the Thynnine wasp Neozeleboria cryptoides (Smith) (Hymenoptera: Tiphiidae), which pollinates the sexually deceptive orchid Chiloglottis trapeziformis Fitzg. (i) When male wasps were given the choice between two female dummies of different sizes and identical amount of synthetic pheromone, they preferentially attempted to copulate with medium-sized dummies over small dummies. (ii) When given the choice between two dummies of identical size but different amounts of pheromone, males preferred the larger amount of pheromone. Larger amounts of pheromone generally attracted more males than smaller amounts. (iii) Orchid flower labella, which mimic a female body, were significantly longer and broader than female wasp bodies, and the flowers also produced on average 10 times more 'pheromone' than females. The evolution and maintenance of these exaggerated mating signals is likely to be mediated by the male pollinator behaviour demonstrated here. (iv) When five dummies were offered simultaneously in a 10 cm circular array, males rarely attempted copulation on more than one dummy during a single visit. This behaviour may foster the evolution or maintenance of clonality in C. trapeziformis, as it will minimize pollen exchange within clones.     

An Analysis of the Effects of Retinoic Acid and Other Retinoids on the Development of Adrenergic Cells from the Avian Neural Crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-transRA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-transRA and 13-cisRA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-transRA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-α, produced an increase in adrenergic cells similar to that seen with all-transRA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-transRA is mediated by RAR-α and possibly RAR-β. To further define the actions of all-transRA on the neural crest we incubated cultures with 5-bromo-2′-deoxyuridine (BrdU) to determine whether all-transRA could affect the rate of proliferation. The results show that while all-transRA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-transRA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.     

Genetic patterns and pollination in Ophrys iricolor and O. mesaritica (Orchidaceae): sympatric evolution by pollinator shift

Ophrys iricolor and O. mesaritica are a pair of morphologically similar, closely related sexually deceptive orchids from the eastern Mediterranean. Ophrys iricolor is known to be pollinated by Andrena morio males and the specific pollinator of Ophrys mesaritica is determined as Andrena nigroaenea. Amplified fragment length polymorphism revealed O. iricolor and O. mesaritica to be genetically intermixed on the whole, although populations of O. iricolor and O. mesaritica in geographical proximity are strongly differentiated, suggesting that specific pollinators locally differentiate these taxa. Based on the available biological data and the system of pollinator attraction operative in Ophrys, we hypothesize that O. mesaritica may have arisen from O. iricolor by pollinator shift and that this is more probable than scenarios invoking hybridization as a result of mispollination by rare, non‐specific flower visitors or specifically attracted insects. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 583–598.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

All-trans-Retinoic Acid Blocks Cell Cycle Progression of Human Ovarian Adenocarcinoma Cells at Late G1

We prepared single cell clones from two ovarian carcinoma cell lines, CA-OV3 and SK-OV3, and analyzed the effect of all-trans-RA treatment on cell division, DNA synthesis, and cell cycle stage distribution of these single cell clones. Our results show that despite the well-known heterogeneous nature of these cell lines, all single cell clones of SK-OV3 cells are resistant to the growth inhibitory effects of all-trans-RA. In contrast, all single cell clones of CA-OV3 cells were growth inhibited by all-trans-RA. However, the extent of growth inhibition did vary somewhat from clone to clone. Additional studies employing flow cytometry showed that all-trans-RA blocked CA-OV3 cell cycle progression in the G₁stage. Finally, all-trans-RA was able to inhibit G₁progression in growth-arrested CA-OV3 cells following stimulation with fetal bovine serum, insulin, IGF-1, or estrogen. Since each of these growth factors is known to act via distinct signal transduction pathways, our results suggest that all-trans-RA blocks G₁progression by targeting a downstream process or event which occurs at a point after the insulin/IGF-1, estrogen, and serum signal transduction pathways converge.     

Evidence of Entrance Sharing and Subterranean Connections in Andrena agilissima (Hymenoptera; Andrenidae)

Andrena agilissima is a bee that nests on sloped or vertical earth walls. Data from 3 years of fieldwork presented here show that this species is communal and uses the same nesting site for many years. On an earth wall on Isola d'Elba (Tuscany, Italy), covered with many bee nest entrances, we studied an aggregation of Andrena agilissima to determine if bees share entrances, if the sharing provides evidence for subterranean connections, and if bees involved in sharing show a division of labor. We marked several females coming out of the same entrance, as well as recapturing the same females using different entrances. No division of labor was observed in our observations.     

Biosynthesis of contact sex pheromone in the female tsetse fly, Glossina morsitans morsitans Westwood

Injection of [2,3 ¹⁴C] sodium succinate into recently emerged, unfed females of Glossina morsitans morsitans resulted in incorporation of radiolabel mainly into surface cuticular alkanes. In vivo experiments with intact flies showed that the distribution of labelled alkanes depended on fly mobility, the legs of unrestrained flies possessing proportionately larger amounts of radioactive hydrocarbon material than those of flies whose legs were tied together with a silk suture. The heads of both restrained and unrestrained flies contained proportionately more material per unit surface area than did any other body part. However, ablation experiments and in vitro incubation showed that the most active incorporation of label into alkanes occurred in the abdomen and that all dorsal abdominal segments were equally active. The ventral abdomen also incorporated label into cuticular alkanes in vitro, but other body parts were apparently less able to do so. The sex pheromone of G. m. morsitans is a trimethyl-substituted alkane, the labelling of which appeared to be in proportion to the relative abundance of its methyl groups among those of the other alkanes of the cuticle following injection of either intact or legless flies. Hence it is proposed that sex pheromone is synthesized along with other cuticular alkanes mainly by cells closely associated with the abdominal cuticle of females and that it is spread over the external surface both by diffusion and by grooming which leads to accumulations of hydrocarbon material on the legs.     

Identification of two components of the female sex pheromone of the sugarcane‐borer Diatraea flavipennella (Lepidoptera: Crambidae)

Analysis by gas chromatography with electroantennographic detection of extracts of pheromone glands derived from calling females of the sugarcane‐borer Diatraea flavipennella revealed two antennally active compounds. These components were identified as (Z)‐9‐hexadecenal (Z9–16:Ald) and (Z)‐11‐hexadecenal (Z11–16:Ald) by comparison of the retention times of the natural compounds and the synthetic compounds supported by two‐dimensional gas chromatography – time‐of‐flight mass spectrometric analysis and the positions of the double bounds in the chains were confirmed from the mass spectral fragmentation patterns of their dimethyldisulphide adducts. The analysis indicated that Z9–16:Ald and Z11–16:Ald were present in the sex pheromone in the proportions 25 : 75. Trace amounts of tetradecanal, hexadecanal, (Z)‐7‐hexadecenal (Z7–16:Ald), (Z)‐9‐hexadecen‐1‐ol and (Z)‐11‐hexadecen‐1‐ol were also found in the extract, but of these only Z9–16:Ald and Z11–16:Ald appeared to be antennally active. Behavioural bioassays demonstrated that a binary blend composed of Z9–16:Ald and Z11–16:Ald in the ratio of 25 : 75 induced a response in D. flavipennella virgin males similar to that elicited by live virgin females or by an hexane extract of the pheromone glands of calling females. Z9–16:Ald and Z11–16:Ald are, therefore, considered to be the major constituents of the female sex pheromone of D. flavipennella.