RESUMO
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.
Assuntos
Antenas de Artrópodes/fisiologia , Cromatografia Gasosa/instrumentação , Mariposas/fisiologia , Álcool Feniletílico/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Animais , Teorema de Bayes , Cromatografia Gasosa/métodos , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Desenho de Equipamento , Feminino , Ionização de Chama/instrumentação , Ionização de Chama/métodos , Método de Monte Carlo , Álcool Feniletílico/análise , Razão Sinal-Ruído , Compostos Orgânicos Voláteis/análiseRESUMO
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.
Assuntos
Besouros/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Sensilas/fisiologia , Atrativos Sexuais/metabolismo , Potenciais de Ação , Animais , Besouros/citologia , Feminino , Masculino , Neurônios Receptores Olfatórios/citologia , Plantas/química , Plantas/metabolismo , Sensilas/citologia , Atrativos Sexuais/química , Comportamento Sexual Animal , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Álcoois/análise , Dípteros/fisiologia , Sesquiterpenos/análise , Atrativos Sexuais/química , Atrativos Sexuais/metabolismo , Animais , Antenas de Artrópodes/fisiologia , Quimiotaxia , Cromatografia Gasosa , Feminino , MasculinoRESUMO
Long-distance flight is crucial for the survival of migratory insects, and disruptions to their flight capacity can have significant consequences for conservation. In this study, we examined how a widely used insecticide, clothianidin (class: neonicotinoid), impacted the flight performance of two species of migratory butterflies, monarchs (Danaus plexippus) and painted ladies (Vanessa cardui). To do this, we quantified the free-flight energetics and tethered-flight velocity and distance of the two species using flow-through respirometry and flight mill assays. Our findings show differential effects of the pesticide on the two species. For painted ladies, we found that clothianidin exposure reduced average free-flight metabolic rates, but did not affect either average velocity or total distance during tethered flight. Other studies have linked low flight metabolic rates with reduced dispersal capacity, indicating that clothianidin exposure may hinder painted lady flight performance in the wild. Conversely, for monarchs, we saw no significant effect of clothianidin exposure on average free-flight metabolic rates but did observe increases in the average velocity, and for large individuals, total distance achieved by clothianidin-exposed monarchs in tethered flight. This suggests a potential stimulatory response of monarchs to low-dose exposures to clothianidin. These findings indicate that clothianidin exposure has the potential to influence the flight performance of butterflies, but that not all species are impacted in the same way. This highlights the need to be thoughtful when selecting performance assays, as different assays can evaluate fundamentally distinct aspects of physiology, and as such may yield divergent results.
RESUMO
Olfactory signals, a major means of communication in insects, travel in the form of turbulent odor plumes. In terrestrial environments, an odor blend emitted from a single point source exists in every strand of the plume, whereas, in confluent plumes from two different odor sources, the strands have some chance of being coincident and comprising a new third odor in those strands. Insects have the ability to detect and interpret necessary olfactory information from individual filamentous odor strands in complex multifilament odor plumes. However, behaviorists have had no way to measure the stimulus situations they are presenting to their temporally acute insect subjects when performing Y-tube olfactometer or confluent pheromone plume wind tunnel assays. We have successfully measured the degree of plume-strand mixing in confluent plumes in a wind tunnel by using a multichannel insect-antenna-based chemosensor. A PC-based computer algorithm to analyze antennal signals from the probe portion of the system performed real-time signal processing and, following a short training session, classified individual odorant/mixture strands at sub-second temporal resolution and a few tens of millimeters of spatial resolution. In our studies, the chemosensor classified a higher frequency of strands of two different odorants emitted from two closely spaced filter papers as being "mixed" when the sources were located only 1 or 2 cm apart than when the sources were 5 or 10 cm apart. These experiments demonstrate the chemosensor's potential to be used for measuring odor stimulus situations in more complex multiple-plume environments.
Assuntos
Lepidópteros/fisiologia , Atrativos Sexuais/farmacologia , Algoritmos , Animais , Masculino , Odorantes , Atrativos Sexuais/químicaRESUMO
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.
Assuntos
Besouros/fisiologia , Sensilas/fisiologia , Olfato , Animais , Besouros/ultraestrutura , Feminino , Masculino , Sensilas/ultraestruturaRESUMO
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.
RESUMO
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.
Assuntos
Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Cromatografia Gasosa/instrumentação , Lepidópteros/química , Odorantes/análise , Órgãos dos Sentidos/química , Animais , Bioensaio/métodos , Cromatografia Gasosa/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Lepidópteros/efeitos dos fármacos , Lepidópteros/fisiologia , Masculino , Reprodutibilidade dos Testes , Órgãos dos Sentidos/efeitos dos fármacos , Órgãos dos Sentidos/fisiologia , Sensibilidade e EspecificidadeRESUMO
Three species of North American heliothine moths were used to determine the level at which interspecific female interference of male attraction to conspecific females occurs. We used live calling females of Heliothis virescens, H. subflexa, and Helicoverpa zea, as lures for conspecific males in a wind tunnel, and then placed heterospecific females on either side of the original species such that the plumes of the three females overlapped downwind. In nearly all combinations, in the presence of heterospecific females, fewer males flew upwind and contacted or courted the source than when only conspecific females were used in the same spatial arrangement. Males did not initiate upwind flight to solely heterospecific female arrangements. Our results show that the naturally emitted pheromone plumes from heterospecific females of these three species can interfere with the ability of females to attract conspecific males when multiple females are in close proximity. However, the fact that some males still located their calling, conspecific females attests to the ability of these male moths to discriminate point source odors by processing the conflicting information from interleaved strands of attractive and antagonistic odor filaments on a split-second basis.