Adaptation of a four-arm olfactometer for behavioural bioassays of large beetles

Summary. We adapted a four-arm olfactometer (55 × 55 × 5.5 cm) for bioassays of large insects and its usefulness was evaluated by testing the responses of three beetles, the palm weevil Rhynchophorus palmarum (L.) (Coleoptera: Curculionidea) and two Dynast scarabs Strategus aloeus (L.) and Oryctes rhinoceros (L.) (Coleoptera: Scarabaeidae) to plant odours and aggregation pheromones. This olfactometer was coupled to a volatile delivery system dispensing highly volatile semiochemicals at constant concentrations. We materialized airflows in the olfactometer by discolouring sensitive papers with acid vapours under various conditions (air straighten by stainless steel grids in inputs and output, 500 ml/min per arm) to visualize air turbulence and to test air-tightness. The volatile delivery system can be used to mix and dispense a broad range of concentrations of semiochemicals in air from diluted aqueous solutions. It was calibrated by measuring the release of the pheromone using solid phase-microextraction (SPME). Dose-response curves to synthetic pheromone were obtained for O. rhinoceros of both sexes. Coupling the volatile delivery system to the fourarm olfactometer provided a system with which the responses of R. palmarum and S. aloeus to their synthetic aggregation pheromones and to natural plant volatiles could be readily observed and studied.     

Development of sol–gel formulations for slow release of pheromones

A new type of dispenser for slow-release of sex pheromones and other semiochemicals was developed based on sol–gel polymers that can be useful for monitoring, mass trapping, and mating disruption in integrated pest management (IPM). Sol–gel matrices exhibit glass characteristics and allow control of the degree of cross-linking during the polymerization process in order to provide an optimal release rate for a particular pheromone. The advantages of sol–gel (silica) matrixes include keeping the added molecules chemically stable and enabling the sol–gel material to be applied in any desired thickness and pheromone quantity, and thereby readily modify release rates. In addition, sol–gels are primarily silica and water that are common in the environment and therefore safe for field dispensing. We developed a method for the entrapment of pheromones in sol–gel matrices that allowed release at an almost constant rate over many days in the field. For example, 2.5 mg (E)-5-decenyl acetate pheromone of peach twig borer, Anarsia lineatella, entrapped in various sol–gel formulations released 14–45 μg/day for up to 28 days. The codling moth (Cydia pomonella) pheromone in sol–gels was used in field tests to capture more codling moth males than unbaited control traps. We describe how the method may be modified to entrap other types of pheromones by making sol–gels with different pore sizes.     

Identification of sex and parasitism via pheromones by the Ozark zigzag salamander

Chemical communication appears to be the primary mode of information transfer among woodland salamanders of the genus Plethodon. These pheromones have been shown to convey a wide variety of information, and are used to establish territorial boundaries. Responses to pheromones can include changes in both behavior and energy consumption. The ability of Ozark zigzag salamanders, Plethodon angusticlavius, to detect the sex of a conspecific through chemical substrate markings has not been assessed. In addition, some populations of P. angusticlavius are naturally infected with the ectoparasitic mite Hannemania eltoni. Parasitism can impact both the behavior and metabolism of the host as well as the behavior of others towards the parasitized individual. We examined whether sex and parasitism of the pheromone donor and receiver influenced behavioral and metabolic responses in P. angusticlavius. In laboratory trials, behavior of P. angusticlavius was influenced by the sex and parasite load of the pheromone donors and receivers. Behavioral responses were also influenced by whether the receiver was in its own territory or in the territory of the pheromone donor. Metabolic responses were not affected by the sex or parasite load of the pheromone donor, but males had higher metabolic rates than females, even after adjusting for differences in body size. Overall, our study indicates that both sex and parasitism can be detected via pheromones in this species, and the nature of the response depends on residency status, sex, and parasite load of the receiver.     

Reversible royalty in worker honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis>) under the queen influence

In most social insects, worker sterility is reversible, and in the absence of the queen, at least some workers develop ovaries and lay male-destined eggs. In the honeybee, reproductive workers also produce queen-characteristic mandibular and Dufour’s pheromones. The evolution of worker sterility is still under debate as to whether it is caused by queen manipulation (queen-control hypothesis) or represents worker fitness maximization (worker-control hypothesis). In this study, we investigated whether worker fertility and royal pheromone production are reversible under the queen influence. To that effect, we induced ovary activation and queen pheromone production in workers by rearing them as queenless (QL) groups. These workers were subsequently reintroduced into queenright (QR) microcolonies for 1 week, and their ovary status and queen pheromone levels were monitored. Workers reintroduced into QR, but not QL colonies, showed a clear regression in ovary development and levels of the queen pheromones. This is the first demonstration that worker sterility and/or fertility is reversible and is influenced by the queen. These results also emphasize the robustness of the coupling between ovary activation and royal pheromone production, as well as lending credence to the queen-control hypothesis. The dynamics of queen pheromone production in QL workers supports the role of Dufour’s gland pheromone as a fertility signal and that of the mandibular gland pheromone in dominance hierarchies.The two authors, Osnat Malka and Shiri Shnieor, contributed equally to this work.     

Human pheromones: have they been demonstrated?

Efforts to collect evidence of human pheromones have focused on three partly overlapping classes of possible human pheromones: (1) axillary steroids, (2) vaginal aliphatic acids, and (3) stimulators of the vomeronasal organ. Examples of each of these classes have been patented for commercial use, and in some cases aggressively marketed, but there is only incomplete evidence supporting any particular claim that a substance acts as a human pheromone. The large axillary scent glands found in humans appear to be well adapted for the production of pheromones, but may actually be used for non-pheromonal odor communication, such as the sharing of information about the immune system. Putative menstrual synchronization within social groups of women and putative acceleration of the menstrual cycle caused by men's odors may suggest the existence of human pheromone systems, but evidence in both cases is still inconclusive.     

Production and transmission of honey bee queen (Apis mellifera L.) mandibular gland pheromone

Summary The social cohesiveness of eusocial insect colonies is maintained primarily through the utilization of pheromones. In this study we quantitatively elucidated the production, secretion, and transmission of 9-keto2(E)-decenoic acid (9-ODA), one of the components of the mandibular gland pheromone of the honey bee queen Apis mellifera; this is the only identified primer pheromone complex in the eusocial insects. Mated queens produce 12–400 g of 9-ODA/day, or between 10% and 170% the average amount found in the glands at any one time. Approximately 0.5 g of 9-ODA is maintained on the body surface of queens by an equilibrium between exudation, internalization, tracking on the comb, and removal by workers. Retinue bees, attending the queen, remove the greatest amount, although the role of the wax as both a sink and a medium for pheromone transfer has been previously underestimated. Only about 1 in 10 retinue workers pick up substantial quantities of pheromone while attending the queen and, within seconds, most of the acquired 9-ODA is found externally on the abdomen, or in the gut. These attendants, also called messenger bees, transfer 9-ODA to other workers, mostly through direct contacts, but also via the wax. A model evaluating the pathways and relative quantities of 9-ODA transferred throughout the nest is presented. As well as being important for a basic understanding of the system, the results have implications for the proper design and use of pheromones in bee management.Offprint requests to: K. Naumann     

Dissecting the mechanisms underlying old male mating advantage in a butterfly

Selection is expected to maximize an individual’s own genetic reward regardless of the potential fitness consequences for its sexual partners, which may cause sexual conflict. Although performance in holometabolous insects typically diminishes with age, old male mating advantage has been documented in a few species. Whether this pattern arises from female preference for older males based on, e.g., pheromone blends (intersexual selection), or from increased eagerness to mate in older compared to younger males is currently debated. We explore the mechanistic basis of old male mating advantage, using a series of experiments including behavioral as well as manipulative approaches, in the tropical butterfly Bicyclus anynana. Consistent with the residual reproductive value hypothesis, old male mating advantage was associated with a greater eagerness to mate, evidenced by a two times higher flying and courting activity in older than in younger males. In contrast, we found only limited support for a contribution of female preference for older males based on pheromone composition, although male sex pheromones clearly do play a role in mating success. Our results suggest that male behavior may play a primary role in old male mating advantage, and that pheromones are likely of secondary importance only. Male mating success was related to higher overall pheromone titers rather than variation in a single component. A dominant importance of male behavior in determining mating success may result in sexual conflict.     

Retinue attraction and ovary activation: responses of wild type and anarchistic honey bees (Apis mellifera) to queen and brood pheromones

In most social insect colonies, workers do not attempt to lay eggs in the presence of a queen. However, in the honey bee (Apis mellifera), a rare phenotype occurs in which workers activate their ovaries and lay large numbers of male eggs despite the presence of a fecund queen. We examined the proximate mechanisms by which this ‘anarchistic’ behaviour is expressed. We tested the effects of brood and queen pheromones on retinue attraction and worker ovary activation using caged worker bees. We found no difference between the anarchistic and wild type queen pheromones in the retinue response elicited in either wild type or anarchistic workers. Further, we found that anarchistic queens produce a pheromone blend that is as effective at inhibiting ovary activation as the wild type queen pheromone. However, anarchistic workers are less inhibited by queen pheromones than their wild type counterparts, in a dose-dependent manner. These results show that the anarchistic phenomenon is not due to changes in the production of queen pheromones, but rather is due in part to a shift in the worker response to these queen-produced signals. In addition, we demonstrate the dose-dependent nature of the effect of queen pheromones on honey bee worker ovary activation.     

Sex pheromones and their potential role in the evolution of reproductive isolation in small ermine moths (Yponomeutidae)

Summary Sex pheromone communication in the nine European species of small ermine moths (Yponomeuta) is reviewed in regard to the potential role of pheromones in the speciation process. Six of the nine species studied (viz.,Y. evonymellus, Y. cagnagellus, Y. padellus, Y. irrorellus, Y. plumbellus, andY. vigintipunctatus) use a mixture of (E)-11-and (Z)-11-tetradecenyl acetate in different ratios as primary pheromone components, with combinations of tetradecyl acetate, (Z)-9-tetradecenyl acetate, (Z)-11-hexadecenyl acetate and the corresponding alcohols of the acetates as additional pheromone components. Analysis of (Z)- to (E)-11-tetradecenyl acetate ratios produced by individual females of these species demonstrated significant variation among females of all species. However, the ranges of ratios produced byY. cagnagellus, Y. irrorellus, andY. plumbellus, sharing the same host-plant species, spindle tree, did not overlap. Niche separation of all six species mentioned required consideration of at least one additional pheromone component or of temporal aspects. The remaining three species,i.e. Y. malinellus, Y. mahalebellus andY. rorellus, have pheromones that differ qualitatively.Biosynthetic routes to the pheromone components identified are proposed on the basis of fatty acid pheromone precursors found in the pheromone glands. A phylogenetic tree for the genus is constructed based on allozyme frequency data and changes in pheromone composition are superimposed on this tree. We suggest that the ancestral ermine moth pheromone is a mixture of (Z)-11- and (E)-11-tetradecenyl acetate and the corresponding alcohols, and a scenario of how present-day patterns evolved is outlined. The pheromone differences among the three species using spindle tree as their host-plant might have evolved throughreproductive character displacement upon secondary contact between populations that had already diverged genetically in allopatry. Pheromone differences within the so-calledpadellus-complex (includingY. cagnagellus, Y. mahalebellus, Y. malinellus, Y. padellus, andY. rorellus) in which species might have originated sympatrically, may have evolved byreinforcing selection as these species still hybridise and produce viable offspring when confined in cages. The role of pheromones in reproductive isolation amongYponomeuta species is emphasised by (1) the function of pheromone components of some of the species as behavioural antagonists to other species, (2) the cross-attraction under experimental conditions between allochronic species with similar pheromones, and (3) the formation of hybrids in the laboratory between species that are isolated in nature by pheromone differences.     

Field bioassays of cerambycid pheromones reveal widespread parsimony of pheromone structures, enhancement by host plant volatiles, and antagonism by components from heterospecifics

Recent research has revealed extensive pheromonal parsimony within the large beetle family Cerambycidae, with closely related species producing the same or very similar pheromone components. This article summarizes research that evaluated attraction of cerambycids to individual pheromone components, blends of pheromone components, and combinations of pheromones with host plant volatiles. Field bioassays were carried out, in collaboration with the Pennsylvania Department of Agriculture and the USDA Cooperative Agricultural Pest Survey program, in 10–25 counties of Pennsylvania over 3 years. A total of 15,438 cerambycids of 134 species were captured, including two exotic species. Semiochemical lures attracted significant numbers of beetles in species of the subfamilies Cerambycinae, Lamiinae, and Spondylidinae, but were not attractive to species in the Lepturinae, Parandrinae, and Prioninae. These experiments reconfirmed the behavioral roles of semiochemicals for a number of species that have been studied previously, and yielded new information about semiochemistry of several species. The host plant volatile α-pinene enhanced attraction of species that were conifer specialists, whereas ethanol enhanced attraction of some species of hardwood specialists. The data suggest that species which share dominant pheromone components avoid cross attraction by differing in seasonal activity period, and by antagonistic effects of minor pheromone components on attraction of heterospecifics. This study further supports the concept that with careful choice of components, multiple pheromones can be deployed as single blends, and paired with host plant volatiles, to maximize the number and taxonomic diversity of cerambycid beetles that are attracted to a single lure, so that the number of different lures that must be deployed can be minimized.     

Colony nutritional status modulates worker responses to foraging recruitment pheromone in the bumblebee Bombus terrestris

Foraging activity in social insects should be regulated by colony nutritional status and food availability, such that both the emission of, and response to, recruitment signals depend on current conditions. Using fully automatic radio-frequency identification (RFID) technology to follow the foraging activity of tagged bumblebees (Bombus terrestris) during 16,000 foraging bouts, we tested whether the cue provided by stored food (the number of full honeypots) could modulate the response of workers to the recruitment pheromone signal. Artificial foraging pheromones were applied to colonies with varied levels of food reserves. The response to recruitment pheromones was stronger in colonies with low food, resulting in more workers becoming active and more foraging bouts being performed. In addition to previous reports showing that in colonies with low food successful foragers perform more excited runs during which they release recruitment pheromone and inactive workers are more prone to leave the nest following nectar influx, our results indicate that evolution has shaped a third pathway that modulates bumblebee foraging activity, thus preventing needless energy expenditure and exposure to risk when food stores are already high. This new feedback loop is intriguing since it involves context-dependent response to a signal. It highlights the integration of information from both forager-released pheromones (signal) and nutritional status (cue) that occurs within individual workers before making the decision to start foraging. Our results support the emerging view that responses to pheromones may be less hardwired than commonly acknowledged. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phenylacetonitrile is a male specific repellent in gregarious desert locusts, Schistocerca gregaria

Summary. The behavioural response of desert locust individuals to adult male volatiles had been investigated in a dynamic Y-T-olfactometer. Synthetic blends of a postulated adult male-produced aggregation pheromone with or without phenylacetonitrile were not attractive to fifth-instar nymphs, young or mature adults of both sexes. Instead phenylacetonitrile had a repellent effect, which was strongest in sexually mature males. The repellent effect of phenylacetonitrile was not modified by the other components of the adult male pheromone bouquet. Even at low concentrations tested the source of the pheromone was avoided. These results are in agreement with recent studies showing that this male specific pheromone is employed by mature gregarious males in sexual behaviour and acts as a courtship inhibiting pheromone. Low concentrations of phenylacetonitrile or the postulated adult male aggregation pheromone blend without phenylacetonitrile may signal the presence of swarm fellows and eventually initiate visually guided congregation behaviour in gregarious locusts. Whether this effect does significantly contribute to swarm cohesion needs further studies.     

Pheromones and colonization: reassessment of the milkweed bug migration model (Heteroptera: Lygaeidae: Lygaeinae)

Summary. Research on insect migration has justifiably emphasized females – the so-called “oogenesis-flight syndrome”– since it is the females that place the eggs into new habitats. The large and small milkweed bugs, Oncopeltus fasciatus and Lygaeus kalmii, respectively, have featured prominently in studies of insect migration and sequestration of host plant toxins for chemical defense. Here we report that males of these species, and males of another well-studied lygaeine (Neacoryphus bicrucis), produce pheromones in glands usually considered to serve only a defensive role in Heteroptera (the metathoracic scent glands), and that these pheromones are exploited by a tachinid parasitoid as a host-finding kairomone. The pheromones are mixtures of C₆ and C₈ saturated and unsaturated esters reminiscent of lepidopteran pheromones, and the key compound of the O. fasciatus pheromone has now been correctly identified as (E)-2,7-octadienyl acetate. It is proposed that the concept of the oogenesis-flight syndrome for these kinds of insects should accommodate the role of males in the migration process. The hypothesis is presented that male-produced pheromones play a significant role in guiding colonization of new habitats in many heteropteran species. In addition, data are presented suggesting that there is a trade-off between the amount of pheromone produced by colonizing males and the host breadth of the species. Received 21 December 1998; accepted 15 February 1999.     

Mixing in the three-dimensional wake of an experimental modelled vehicle

Experimental results on tracer gas diffusion within the near wake of a simplified model car (Ahmed model with a rear slant angle of 25°) are presented. Pollutant emission is simulated using heated air injected through a small pipe at one side of the model base. Fine cold wire thermometry is used to measure instantaneous temperature excess and variance of temperature gradient in the near wake. Measurements of the three mean velocity components were made using a laser Doppler anemometers system. Characteristics of the mean and fluctuating temperature fields, time-averaged flow streamlines and scalar dissipation measurements are presented and discussed. The local mixing time scale is determined from the measured mean dissipation rate of temperature variance. Its value shows that micromixing is not a limiting phenomenon for chemical reactions in the near wake.     

Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers

Several insect pheromones are multifunctional and have both releaser and primer effects. In honey bees (Apis mellifera), the queen mandibular pheromone (QMP) and e-beta-ocimene (eβ), emitted by young worker larvae, have such dual effects. There is increasing evidence that these multifunctional pheromones profoundly shape honey bee colony dynamics by influencing cooperative brood care, a fundamental aspect of eusocial insect behavior. Both QMP and eβ have been shown to affect worker physiology and behavior, but it has not yet been determined if these two key pheromones have interactive effects on hypopharyngeal gland (HPG) development, actively used in caring of larvae, and ovary activation, a component of worker reproductive physiology. Experimental results demonstrate that both QMP and eβ significantly suppress ovary activation compared to controls but that the larval pheromone is more effective than QMP. The underlying reproductive anatomy (total ovarioles) of workers influenced HPG development and ovary activation, so that worker bees with more ovarioles were less responsive to suppression of ovary activation by QMP. These bees were more likely to develop their HPG and have activated ovaries in the presence of eβ, providing additional links between nursing and reproductive physiology in support of the reproductive ground plan hypothesis.     

Attraction of field-flying aphid males to synthetic sex pheromone

Summary Transparent plastic water traps, baited with the synthetic aphid sex pheromone components (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol or control lures, were placed in four semi-sheltered field sites at a height of 1.1 m. Although males of twenty-one aphid species were collected from the water traps, onlySitobion fragariae (Walker) was abundant. In total, 8 pheromone traps produced 102 males compared with only 10 males in 8 control traps. The sex pheromone released by sexual femaleS. fragariae was identified as the nepetalactone used in the lures. Aphid sex pheromones may be more species-specific than previously thought and the presence of a host plant is not essential for males to locate sexual females.     

A simulation model of population dynamics of the codling moth, Cydia pomonella

A simulation model has been developed that predicts numbers and phenology of a population of codling moth, Cydia pomonella (L.), in an apple orchard. The model is a general insect population model based on the interative-cohort technique. It operates at two time scales: a fine time scale (1 h) for temperature-dependent physiological processes, and a coarse time scale (1 day) for population processes. The population is divided into a specifiable number of stages, and each stage is described by four process functions, which may be of any convenient mathematical form, and may differ among stages. Each stage is divided into cohorts of individuals born or emerged on the same day, and individuals within a cohort are considered probabilistically identical. The model simulates the processes of development, transition among stages, and mortality by using probability distributions representing these processes, and incorporates the effects of pesticides on mortality of the insect. Model output was evaluated by comparison with records of pheromone trap catches of codling moths in commercial apple orchards in North Carolina. The model predicts timing of the first spring flight well, depending on the initial age distribution used. Time between peaks of numbers of adults in the model is about 15 days longer than the observed period between flight peaks in orchards. Sensitivity analysis indicates that this discrepancy may be related to differences between measured ambient temperature and tree canopy temperature. The sensitivities of numbers of insects produced by the model, and timing of peaks in numbers present were determined for each of the parameters in the model. The parameters with greatest effect on the model output were those which control the locations of developmental rate functions and survival functions on the temperature scale. In the model, pesticides had a much larger effect on numbers of adults present than records of moths caught in pheromone traps indicate actually occurred, suggesting that moths caught in traps in commercial orchards where effective pesticides are applied may be largely immigrants.     

Mixed messages across multiple trophic levels: the ecology of bark beetle chemical communication systems

Summary. Chemical, physiological and behavioral components of pheromone communication have been described for a number of bark beetle species, yet our understanding of how these signals function under natural conditions remains relatively limited. Development of ecologically based models is complicated by the multiple functions and sources of variability inherent in bark beetle semiochemistry. This discussion addresses four ecological issues of chemical signaling in bark beetles: the effects of aggregation on individual fitness, the possibility of cheating, how plants can defend themselves against herbivores that employ aggregation pheromones, and the implications of variability in chemical communication systems to predator avoidance. An analysis of published data from thirteen scolytid – conifer systems indicates that the net benefit and optimal colonization density vary with host condition and beetle species. When beetles attack live trees, the benefit of cooperative host procurement exceeds losses due to competition for the limited substrate, at least up to moderate densities. When beetles colonize dead tissue, however, the effect of subsequently arriving beetles on initial colonizers is almost entirely negative. This suggests that aggregation originated as exploitation of senders, but evolved into manipulation of receivers. It is also proposed that the optimal colonization density which typifies each species or population may offer a more objective and less value–laden index of behavior than current labels such as “aggressiveness”. Beetles can maximize the relative benefits of group attack by incorporating instantaneous measures of host resistance into their colonization behavior, and by adjusting oviposition with colonization density. This system may provide opportunities for cheating. However a number of factors may select against a fixed strategy of cheating, including the linkage between tree allelochemistry and beetle semiochemistry, the reduced quality of substrate available to late arrivers, the short adult lifespans of most bark beetles, differential exposure to some predators, the difficulty of locating signalers during extensive endemic periods, and the low costs incurred during host assessment. However, the possibility that beetles employ flexible, density – dependent strategies deserves heightened attention. The ability of bark beetles to collectively exhaust host defenses poses a particular problem for plant defense. It is argued here that the ideal defense should include both direct resistance mechanisms against invading beetles, and indirect mechanisms that inhibit chemical communication. Evidence for the latter mechanism is explored. The ability of predators to efficiently exploit aggregation pheromones as kairomones in prey finding poses significant risk to bark beetles. It is proposed that minor alterations in pheromone components may provide colonizers with partial escape from such natural enemies while maintaining intraspecific functionality. Traditional interpretations emphasized the fidelity and consistency of pheromones, but under natural conditions chemical signals are modified by unpredictable features of the biotic and abiotic environment. Although we typically view variation in pheromonal signals as experimental noise or simple deviations from a population norm, such variation may reflect evolutionary dynamics. Complex ecological interactions may impose trade-offs between the clarity versus diversity of their signals. Received 3 July 2000; accepted 8 January 2001     

Population differentiation in female sex pheromone and male preferences in a solitary bee

Population differentiation in female mating signals and associated male preferences can drive reproductive isolation among segregated populations. We tested this assumption by investigating intraspecific variation in female sex pheromone and associated male odour preferences among distant populations in the solitary bee Colletes cunicularius (L.) by using quantitative gas chromatography and by performing field bioassays with synthetic blends of key sex pheromone compounds. We found significant differences in sex pheromone blends among the bee populations, and the divergence in odour blends correlated positively with geographic distance, suggesting that genetic divergence among distant populations can affect sex pheromone chemistry. Our behavioural experiments, however, demonstrate that synthetic copies of allopatric female sex pheromones were cross-attractive to patrolling males from distant populations, making reproductive isolation by non-recognition of mating signals among populations unlikely. Our data also show that patrolling male bees from different populations preferred odour types from allopatric populations at the two sites of bioassays. These male preferences are not expected to select for changes in the female sex pheromone, but may influence the evolution of floral odour in sexually deceptive orchids of the genus Ophrys that are pollinated by C. cunicularius males.