Pheromone Odorant Receptor Responses Reveal the Presence of a Cryptic, Redundant Sex Pheromone Component in the European Corn Borer, Ostrinia nubilalis.

Two odorant receptors (ORs), OnubOR3 and OnubOR6, in the sex pheromone communication systems of E- and Z-strain European corn borers, Ostrinia nubilalis, were broadly receptive to analogs of their pheromone components. In addition to responding to their natural 14-carbon pheromone components, (Z)-11- and (E)-11-tetradecenyl acetates (Z11- and E11-14:OAc), these pheromone ORs responded to the longer-chain compounds, (Z)-11- and (E)-11-hexadecenyl acetate (Z11- and E11-16:OAc). Z11-16:OAc is a pheromone gland constituent of E-strain O. nubilalis females in Europe but has not previously been shown to have behavioral activity to males. Here, we demonstrate that Z11-16:OAc evokes high levels of upwind flight and source location in E-strain males when substituted for Z11-14:OAc (minor component) in the E-strain blend. Since Z11-16:OAc is found in the gland and has behavioral activity when Z11-14:OAc is missing, then it should be classified as a cryptic, redundant minor pheromone component in E-strain O. nubilalis. The opposite geometric isomer, E11-16:OAc, also functions in Z-strain O. nubilalis, substituting behaviorally for the E11-14:OAc minor component, but has not been found in Z-strain female glands. Single-sensillum recordings showed that sensory neurons of E- and Z-strain male antennae expressing OnubOR3 and OnubOR6 produced responses to these hexadecenyl acetates similar to those evoked by the natural (tetradecenyl acetate) pheromone components. We postulate that the wide responsiveness of these two ORs to the 16-carbon acetates could be a preadaptation for O. nubilalis to use these compounds as minor components in lieu of the respective 14-carbon acetates. Alternatively, the responsiveness of OnubOR3 to E11-16:OAc and OnubOR6 to Z11-16:OAc could represent a vestigial state of these receptors, with the 16-carbon acetates having previously acted as functional minor components in an ancestral blend.

Choice of Laboratory Tissue Homogenizers Matters When Recovering Nucleic Acid From Medically Important Ticks.

Ticks can vector and transmit many pathogens and pose a serious human health threat throughout the world. After collection, many diagnostic laboratories must mechanically disrupt tick specimens for diagnostic testing and research purposes, but few studies have evaluated how well-commercial tissue homogenizers perform this task. We evaluated four commercially available tissue homogenizers: The Bead Ruptor 24 Elite, the Bullet Blender Storm, the gentleMACS Dissociator, and the Precellys 24. We quantitatively compared maceration level, nucleic acid quality, quantity, amplification, and DNA shearing to determine which machines performed the best. The Bead Ruptor 24 Elite had the highest overall score when disrupting a single, uninfected adult Amblyomma americanum (Linnaeus) (Ixodida: Ixodidae) and performed well in follow-on tests including disrupting individual juvenile samples and detecting pathogens from infected samples.

Labial and maxillary palp recordings of the Asian longhorned beetle, Anoplophora glabripennis, reveal olfactory and hygroreceptive capabilities.

Electrophysiological recordings from the labial and maxillary palps of the Asian longhorned beetle, Anoplophora glabripennis, revealed their ability to detect several volatile chemicals, including water vapor and acetic acid. The results indicate that these appendages may play a large role in this beetle's assessment of its immediate environment. A. glabripennis is a highly destructive, invasive pest that feeds preferentially on maple - but accepts many other tree species - in North America, warranting USDA quarantine zones and an eradication program. While control and sampling techniques are being developed for this insect, a better understanding of its sensory capabilities is helpful. Electropalpograms (EPGs) revealed that both the maxillary and labial palps are highly sensitive to changes in humidity, indicating the presence of hygroreceptors and the likely important role of humidity in such things as feeding and finding water or oviposition sites. Strong EPG responses to a narrow set of volatile chemicals indicate that olfactory sensory neurons (OSNs) on the palps may be tuned to a small number of volatile compounds. The types of odorant molecules eliciting responses indicate that there are likely both odorant receptors (ORs) as well as ionotropic receptors (IRs) expressed on the OSNs, enabling palp OSNs to be able to respond to acids and aldehydes such as acetic acid and butyraldehyde. There were no significant EPG responses to this species' trail-sex pheromone components, which may indicate that the trail pheromone is primarily perceived via gustatory receptors contacting the substrate. These results indicate that the palps have a role in the beetle's assessment of its immediate environment underfoot, and that the sampling of surface odors and humidity via mouth parts may be important to this species' success.

Olfactory Sensory Neurons of the Asian Longhorned Beetle, Anoplophora glabripennis, Specifically Responsive to its two Aggregation-Sex Pheromone Components.

We performed single-sensillum recordings from male and female antennae of the Asian longhorned beetle, Anoplophora glabripennis, that included as stimuli the two components of this species' aggregation-sex pheromone in addition to various general odorants. We compared the aggregation-sex-pheromone-component responses of olfactory sensory neurons (OSNs) to those of OSNs that responded to a variety of plant-related odorants. In the smooth-tipped, tapered, trichoid sensilla on the most distal antennal flagellomeres nos. 10 or 11 of both males and females, we found OSNs with high-amplitude action potentials that were tuned to the aldehyde and alcohol pheromone components and that did not respond to various plant-related volatiles. Because this OSN type responded to both the alcohol and aldehyde components it cannot be considered to be specifically tuned to either component. These large-spiking OSNs were co-compartmentalized in these sensilla with a second, smaller-spiking OSN responding to plant-related volatiles such as geraniol, citronellal, limonene, 1-octanol, nerol and citral. The large-spiking OSNs thus appear to be a type that will be involved in aggregation-sex pheromone pathways targeting a specific glomerulus in the antennal lobe and in generating pheromone-related behavioral responses in A. glabripennis. In other sensilla located in these distal antennal flagellomeres as well as those located more proximally, i.e., mid-length along the antenna on flagellomere nos. 4-7, we found OSNs in blunt-tipped basiconic sensilla that were responsive to other plant-related volatiles, especially the terpenoids, (E,E)-alpha farnesene, (E)-ß-farnesene, ß-caryophyllene, and eugenol. Some of these terpenoids have been implicated in improving attraction to pheromone-baited traps. Some of these same OSNs responded additionally to either of the two sex pheromone components, but because these OSNs also responded to some of the above plant volatiles as shown by cross-adaptation experiments, these OSNs will not be the types that convey sex-pheromone-specific information to the antennal lobe.

Increasing Signal-to-Noise Ratio in Gas Chromatography - Electroantennography Using a Deans Switch Effluent Chopper.

Gas-chromatography-electroantennographic detection (GC-EAD) is a technique used in the identification of volatile organic compounds (VOCs), such as pheromones and plant host odors, which are physiologically relevant to insects. Although pheromones often elicit large EAD responses, other behaviorally relevant odors may elicit responses that are difficult to discern from noise. Lock-in amplification has long been used to reduce noise in a wide range of applications. Its utility when incorporated with GC-EAD was demonstrated previosuly by chopping (or pulsing) effluent-laden air that flowed over an insect antenna. This method had the disadvantage that it stimulated noise-inducing mechanoreceptors and, in some cases, disturbed the electrochemical interfaces in a preparation, limiting its performance. Here, the chopping function necessary for lock-in amplification was implemented directly on the GC effluent using a simple Deans switch. The technique was applied to excised antennae from female Heliothis virescens responding to phenethyl alcohol, a common VOC emitted by plants. Phenethyl alcohol was always visible and quantifiable on the flame ionization detector (FID) chromatogram, allowing the timing and amount of stimulus delivered to the antennal preparation to be measured. In our new chopper EAG configuration, the antennal preparation was shielded from air currents in the room, further reducing noise. A dose-response model in combination with a Markov-chain monte-carlo (MCMC) method for Bayesian inference was used to estimate and compare performance in terms of error rates involved in the detection of insect responses to GC peaks visible on an FID detector. Our experiments showed that the predicted single-trial phenethyl alcohol detection limit on female H. virescens antennae (at a 5.0% expected error rate) was 140,330 pg using traditional EAG recording methods, compared to 2.6-6.3 pg (5th to the 95th percentile) using Deans switch-enabled lock-in amplification, corresponding to a 10.4-12.7 dB increase in signal-to-noise ratio.

Odorant receptors and antennal lobe morphology offer a new approach to understanding olfaction in the Asian longhorned beetle.

The Asian longhorned beetle Anoplophora glabripennis (Motchulsky) is an exotic forest pest that has repeatedly invaded North America and Europe from Asia, and has the potential to kill millions of trees and cause billions of dollars in damage. Traps baited with an attractive mixture of volatile organic compounds from hosts have been of limited success in monitoring invasion sites. We propose that lures might be improved through studying the olfactory system of adult beetles, especially the gene family of odorant receptors (ORs) and the structure of the antennal lobes of the brain. Here, we report identification of 132 ORs in the genome of A. glabripennis (inclusive of one Orco gene and 11 pseudogenes), some of which are orthologous to known pheromone receptors of other cerambycid beetles. We also identified three ORs that are strongly biased toward expression in the female transcriptome, and a single OR strongly biased toward males. Three-dimensional reconstruction of the antennal lobes of adults suggested a male-specific macroglomerulus and several enlarged glomeruli in females. We predict that functional characterization of ORs and glomeruli will lead to identification of key odorants in the life history of A. glabripennis that may aid in monitoring and controlling future invasions.

Chopper-modulated gas chromatography electroantennography enabled using high-temperature MEMS flow control device.

We report the design, fabrication and characterization of a microelectromechanical systems (MEMS) flow control device for gas chromatography (GC) with the capability of sustaining high-temperature environments. We further demonstrate the use of this new device in a novel MEMS chopper-modulated gas chromatography-electroantennography (MEMS-GC-EAG) system to identify specific volatile organic compounds (VOCs) at extremely low concentrations. The device integrates four pneumatically actuated microvalves constructed via thermocompression bonding of the polyimide membrane between two glass substrates with microstructures. The overall size of the device is 32 mm×32 mm, and it is packaged in a 50 mm×50 mm aluminum housing that provides access to the fluidic connections and allows thermal control. The characterization reveals that each microvalve in the flow control chip provides an ON to OFF ratio as high as 1000:1. The device can operate reliably for more than 1 million switching cycles at a working temperature of 300 °C. Using the MEMS-GC-EAG system, we demonstrate the successful detection of cis-11-hexadecenal with a concentration as low as 1 pg at a demodulation frequency of 2 Hz by using an antenna harvested from the male Helicoverpa Virescens moth. In addition, 1 µg of a green leafy volatile (GLV) is barely detected using the conventional GC-EAG, while MEMS-GC-EAG can readily detect the same amount of GLV, with an improvement in the signal-to-noise ratio (SNR) of ~22 times. We expect that the flow control device presented in this report will allow researchers to explore new applications and make new discoveries in entomology and other fields that require high-temperature flow control at the microscale.

Attraction, Oviposition and Larval Survival of the Fungus Gnat, Lycoriella ingenua, on Fungal Species Isolated from Adults, Larvae, and Mushroom Compost.

We previously showed that the females of the mushroom sciarid, Lycoriella ingenua (Dufour, 1839) (Diptera: Sciaridae), one of the most severe pests of the cultivated white button mushroom, Agaricus bisporus (J.E. Lange) Emil J. Imbach (Agaricales: Agaricaceae), are attracted to the mushroom compost that mushrooms are grown on and not to the mushrooms themselves. We also showed that females are attracted to the parasitic green mold, Trichoderma aggressivum. In an attempt to identify what is in the mushroom compost that attracts female L. ingenua, we isolated several species of fungi from adult males and females, third instar larvae, and mushroom compost itself. We then analyzed the attraction of females to these substrates using a static-flow two choice olfactometer, as well as their oviposition tendencies in another type of assay under choice and no-choice conditions. We also assessed the survival of larvae to adulthood when first instar larvae were placed on each of the isolated fungal species. We found that female flies were attracted most to the mycoparasitic green mold, T. aggressivum, to Penicilium citrinum isolated from adult female bodies, and to Scatylidium thermophilium isolated from the mushroom compost. Gravid female flies laid the most eggs on T. aggressivum, Aspergillus flavus isolated from third instar larval frass, Aspergillus fumigatus isolated from adult male bodies, and on P. citrinum. This egg-laying trend remained consistent under no-choice conditions as females aged. First instar larvae developed to adulthood only on S. thermophilium and Chaetomium sp. isolated from mushroom compost, and on P. citrinum. Our results indicate that the volatiles from a suite of different fungal species act in tandem in the natural setting of mushroom compost, with some first attracting gravid female flies and then others causing them to oviposit. The ecological context of these findings is important for creating an optimal strategy for using possible semiochemicals isolated from these fungal species to better monitor and control this pestiferous mushroom fly species.

Differences in spectral selectivity between stages of visually guided mating approaches in a buprestid beetle.

Spectral mating preferences were examined in male Agrilus angustulus (Buprestidae: Coleoptera), a member of a taxon known for its high species diversity and striking metallic coloration. The spectral emission profile of a typical A. angustulus female displays low chroma, broadly overlapping that of the green oak leaves they feed and rest upon, while also including longer wavelengths. To pinpoint behaviorally significant spectral regions for A. angustulus males during mate selection, we observed their field approaches to females of five Agrilus planipennis color morphs that have greater chroma than the normal conspecific female targets. Agrilus angustulus males would initially fly equally frequently toward any of the three longest wavelength morphs (green, copper and red) whose spectral emission profiles all overlap that of typical A. angustulus females. However, they usually only completed approaches toward the two longest wavelength morphs, but not the green morphs. Thus, spectral preference influenced mate selection by A. angustulus males, and their discrimination of suitable targets became greater as these targets were approached. This increasing spectral discrimination when approaching targets may have evolved to allow female emissions to remain somewhat cryptic, while also being visible to conspecifics as distinct from the background vegetation and heterospecific competitors.

Interaction of Visual and Chemical CUES in Promoting Attraction of Agrilus planipennis.

Female emerald ash borers, Agrilus planipennis (Coleoptera: Buprestidae), emit a macrocyclic lactone, (Z)-3-dodecen-12-olide, that increases field trap captures on large-panel prism traps when co-emitted with the green leaf volatile (Z)-3-hexenol. We assessed attraction to these compounds by using visual decoy-baited branch traps, which attract males by mimicking a living female resting upon a leaf. Pairs of branch traps, with and without visual decoy beetles, were placed on green ash, Fraxinus pennsylvanica, trees, which were assigned different odor treatments: 1) no odor, 2) (Z)-3-hexenol alone, and 3) (Z)-3-hexenol-plus-lactone. Male captures were positively affected by the presence of decoys and the emission of either (Z)-3-hexenol or (Z)-3-hexenol plus lactone. The decoy-baited traps with the combination of (Z)-3-hexenol plus lactone caught more males than any other treatment. Greater male captures were associated with continuing captures later in the season, suggesting that decoy and odor attractants remain attractive throughout the flight period. Female captures were not affected by the visual decoys, but odors did influence captures, with the (Z)-3-hexenol plus lactone treatment catching the greatest number of females. The rare female trap captures were negatively correlated with the more common male captures on the odorless and (Z)-3-hexenol-baited traps, but were not correlated with male captures when the lactone was added. Thus, in the absence of the lactone, the visual signal of other conspecifics can inhibit female attraction. However, the pheromone attracts both sexes independently of the visual signal on the trap.

Isolation of a Female-Emitted Sex Pheromone Component of the Fungus Gnat, Lycoriella ingenua, Attractive to Males.

Lycoriella ingenua Dufour (Diptera: Sciaridae) is acknowledged as the major pest species of the white button mushroom, Agaricus bisporus, throughout the world. Components of the female-produced sex pheromone of this species were identified previously as C15-C18 n-alkanes, with the major component n-heptadecane, and shown to be attractive to L. mali. However, a subsequent report could not repeat this work. We reinvestigated the sex pheromone of this species by confirming that virgin females were attractive to males in a Y-tube bioassay and by collection of extracts from virgin females. Extracts were analyzed by gas chromatography coupled to electroantennographic detection, and by the less widely-used technique of gas chromatography coupled to a behavioral bioassay to detect compounds causing wing-fanning and copulatory abdomen curling in males. A single, behaviorally-active pheromone component was isolated and characterized by gas chromatography coupled to mass spectrometry. This component was definitively not n-heptadecane or any of the other C15-C19 n-alkanes reported previously, but is proposed to be a sesquiterpene alcohol having analytical characteristics that closely matched those of reference germacradienols.

Neurophysiological mechanisms underlying sex- and maturation-related variation in pheromone responses in honey bees (Apis mellifera).

In the honey bee (Apis mellifera), social organization is primarily mediated by pheromones. Queen-produced 9-oxo-2-decenoic acid (9-ODA) functions as both a social and sex pheromone, eliciting attraction in both female workers and male drones, but also affecting other critical aspects of worker physiology and behavior. These effects are also maturation related, as younger workers and sexually mature drones are most receptive to 9-ODA. While changes in the peripheral nervous system drive sex-related differences in sensitivity to 9-ODA, the mechanisms driving maturation-related shifts in receptivity to 9-ODA remain unknown. Here, we investigate the hypothesis that changes at the peripheral nervous system may be mediating plastic responses to 9-ODA by characterizing expression levels of AmOR11 (the olfactory receptor tuned to 9-ODA) and electrophysiological responses to 9-ODA. We find that receptor expression correlates significantly with behavioral receptivity to 9-ODA, with nurses and sexually mature drones exhibiting higher levels of expression than foragers and immature drones, respectively. Electrophysiological responses to 9-ODA were not found to correlate with behavioral receptivity or receptor expression, however. Thus, while receptor expression at the periphery exhibits a level of plasticity that correlates with behavior, the mechanisms driving maturation-dependent responsiveness to 9-ODA appear to function primarily in the central nervous system.

Malaria mosquitoes host-locate and feed upon caterpillars.

Adult female mosquitoes need blood to develop their eggs and both sexes use nectar and honeydew as carbohydrate resources for flight, survival and to enhance reproduction. However, there are also a few reports in the literature of mosquitoes feeding on haemolymph of soft-bodied insects such as caterpillars. The frequency and significance of this entomophagous behavior is not well understood, but is thought to be a vestige of ancestral feeding behavior or an opportunistic behavior that has evolved over time. In our current paper we investigated the extent to which the malaria mosquito, Anopheles stephensi, is attracted to, and can successfully feed on, larvae of two common moth species, Manduca sexta and Heliothis subflexa. Using y-tube olfactometer assays we found that female An. stephensi readily flew upwind to and landed on the caterpillars of both moth species. The nature of the volatile cues used in host location remains unclear but respirometer studies suggest a possible role of CO2. Laboratory cage assays further showed that the female mosquitoes were able to actively feed on moth larvae and gain sufficient nutritional benefit to influence survival. The extent to which such an opportunistic behavior occurs in the field has yet to be explored but our results suggest that this haemolymph feeding behavior could play a role in malaria mosquito life history and could provide a novel mechanism for horizontal transmission of pathogens and other micro-organisms between hosts.

Bioreplicated visual features of nanofabricated buprestid beetle decoys evoke stereotypical male mating flights.

Recent advances in nanoscale bioreplication processes present the potential for novel basic and applied research into organismal behavioral processes. Insect behavior potentially could be affected by physical features existing at the nanoscale level. We used nano-bioreplicated visual decoys of female emerald ash borer beetles (Agrilus planipennis) to evoke stereotypical mate-finding behavior, whereby males fly to and alight on the decoys as they would on real females. Using an industrially scalable nanomolding process, we replicated and evaluated the importance of two features of the outer cuticular surface of the beetle's wings: structural interference coloration of the elytra by multilayering of the epicuticle and fine-scale surface features consisting of spicules and spines that scatter light into intense strands. Two types of decoys that lacked one or both of these elements were fabricated, one type nano-bioreplicated and the other 3D-printed with no bioreplicated surface nanostructural elements. Both types were colored with green paint. The light-scattering properties of the nano-bioreplicated surfaces were verified by shining a white laser on the decoys in a dark room and projecting the scattering pattern onto a white surface. Regardless of the coloration mechanism, the nano-bioreplicated decoys evoked the complete attraction and landing sequence of Agrilus males. In contrast, males made brief flying approaches toward the decoys without nanostructured features, but diverted away before alighting on them. The nano-bioreplicated decoys were also electroconductive, a feature used on traps such that beetles alighting onto them were stunned, killed, and collected.

Malaria mosquitoes attracted by fatal fungus.

Insect-killing fungi such as Beauveria bassiana are being evaluated as possible active ingredients for use in novel biopesticides against mosquito vectors that transmit malaria. Fungal pathogens infect through contact and so applications of spores to surfaces such as walls, nets, or other resting sites provide possible routes to infect mosquitoes in and around domestic dwellings. However, some insects can detect and actively avoid fungal spores to reduce infection risk. If true for mosquitoes, such behavior could render the biopesticide approach ineffective. Here we find that the spores of B. bassiana are highly attractive to females of Anopheles stephensi, a major anopheline mosquito vector of human malaria in Asia. We further find that An. stephensi females are preferentially attracted to dead and dying caterpillars infected with B. bassiana, landing on them and subsequently becoming infected with the fungus. Females are also preferentially attracted to cloth sprayed with oil-formulated B. bassiana spores, with 95% of the attracted females becoming infected after a one-minute visit on the cloth. This is the first report of an insect being attracted to a lethal fungal pathogen. The exact mechanisms involved in this behavior remain unclear. Nonetheless, our results indicate that biopesticidal formulations comprising B. bassiana spores will be conducive to attraction and on-source visitation by malaria vectors.

'Manipulation' without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.

Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this 'manipulation phenotype' to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour.

Chopper-stabilized gas chromatography-electroantennography: Part I. background, signal processing and example.

A new method that can improve gas-chromatography-electroantennographic detection (GC-EAD) by orders of magnitude through a technique known as chopper stabilization combined with matched filtering in colored noise is presented. The EAD is a physiological recording from the antenna of an insect which can be used to find compounds in the GC effluent that the antenna is able to detect, having important applications for pest control and understanding of chemical communication in nature. The new method is demonstrated with whole-animal male Helicoverpa zea antennal preparations for detection of major pheromone component (cis-11-hexadecenal) and compared to results obtained using traditional EAD recording techniques. Results indicate that chopper stabilization under these circumstances can increase odorant detection performance by a factor of approximately 10(4) over traditional methods. The time course of the response of the antenna is also better resolved under chopped conditions. Although the degree of improvement is expected to vary with insect species, odor, and insect preparation, under most circumstances a more sensitive and robust GC-EAD instrument will result from the application of this technique.