Optimizing the Point-Source Emission Rates and Geometries of Pheromone Mating Disruption Mega-Dispensers.

High-emission-rate "mega-dispensers" have come into increasing use for sex pheromone mating disruption of moth pests over the past two decades. These commercially available dispensers successfully suppress mating and reduce crop damage when they are deployed at very low to moderate densities, ranging from 1 to 5/ha to 100-1000/ha, depending on the dispenser types and their corresponding pheromone emission rates. Whereas traditionally the emission rates for successful commercial mating disruption formulations have been measured in terms of amounts (usually milligram) emitted by the disruptant application per acre or hectare per day, we suggest that emission rates should be measured on a per-dispenser per-minute basis. In addition we suggest, because of our knowledge concerning upwind flight of male moths being dependent on contact with pheromone plume strands, that more attention needs to be paid to optimizing the flux within plume strands that shear off of any mating disruption dispenser's surface. By measuring the emission rates on a per-minute basis and measuring the plume strand concentrations emanating from the dispensers, it may help improve the ability of the dispensers to initiate upwind flight from males and initiate their habituation to the pheromone farther downwind than can otherwise be achieved. In addition, by optimizing plume strand flux by paying attention to the geometries and compactness of mating disruption mega-dispensers may help reduce the cost of mega-dispenser disruption formulations by improving their behavioral efficacy while maintaining field longevity and using lower loading rates per dispenser.

Working range of stimulus flux transduction determines dendrite size and relative number of pheromone component receptor neurons in moths.

We are proposing that the "relative" abundances of the differently tuned pheromone-component-responsive olfactory receptor neurons (ORNs) on insect antennae are not a result of natural selection working to maximize absolute sensitivity to individual pheromone components. Rather, relative abundances are a result of specifically tuned sensillum-plus-ORN units having been selected to accurately transduce and report to the antennal lobe the maximal ranges of molecular flux imparted by each pheromone component in every plume strand. To not reach saturating stimulus flux levels from the most concentrated plume strands of a pheromone blend, the dendritic surface area of the ORN type that is tuned to the most abundant component of a pheromone blend is increased in dendritic diameter in order to express a greater number of major pheromone component-specific odorant receptors. The increased ability of these enlarged dendrite, major component-tuned ORNs to accurately report very high flux of its component results in a larger working range of stimulus flux able to be accurately transduced by that type of ORN. However, the larger dendrite size and possibly other high-flux adjustments in titers of pheromone-binding proteins and degrading enzymes cause a decrease in absolute sensitivity to lower flux levels of the major component in lower concentration strands of the pheromone blend. In order to restore the ability of the whole-antenna major pheromone component-specific channel to accurately report to its glomerulus the abundance of the major component in lower concentration strands, the number of major component ORNs over the entire antenna is adjusted upward, creating a greater proportion of major component-tuned ORNs than those tuned to minor components. Pheromone blend balance reported by the whole-antennal major and minor component channels in low plume-flux strands is now restored, and the relative fluxes of the 2 components occurring in both low- and high-flux strands are thereby accurately reported to the component-specific glomeruli. Thus, we suggest that the 2 phenomena, dendrite size and relative numbers of differentially tuned ORNs are linked, and both are related to wide disparities in molecular flux ranges occurring for the more abundant and less abundant components in the pheromone blend plume strands.

Evolution of male pheromones in moths: reproductive isolation through sexual selection?

Central to our understanding of the species concept is knowledge of the nature and evolution of reproductive isolating mechanisms. The once widely accepted model of Dobzhansky, which holds that isolation evolves through selection against hybrids of differentially adapted populations, is now largely rejected. This rejection is due to both theoretical difficulties and a paucity of examples of the predicted pattern of reproductive character displacement. From a survey of five families of Lepidoptera, entailing more than 800 species, evidence is given that male courtship pheromones have evolved within the context of sexual isolation as an adaptive response to mating mistakes between differentially adapted populations; however, distinct from the natural selection model of Dobzhansky, this report suggests the mechanism for change to be sexual selection.

Pheromone source location by flying moths: a supplementary non-anemotactic mechanism.

After the wind was stopped in an insect flight tunnel, male oriental fruit moths continued to fly in zigzag fashion along a stationary pheromone plume. Their lateral excursions from the time-averaged pheromone plume were no greater without wind than in wind of 38 centimeters per second. When the pheromone plume was removed and the wind stopped, males initiated wider track reversals when they reached the pheromone-free area in still air than they had made while in the pheromone plume. This non-anemotactic mechanism of maintaining plume contact-possibly a special kind of klinotaxis-when coupled with the orthokinetic retinal velocity of apparent ground pattern motion, allowed males to reach the pheromone source area from 1 to 2 meters away without wind.

Close-range attraction of female oriental fruit moths to herbal scent of male hairpencils.

A blend of ethyl trans-cinnamate, methyl 2-epijasmonate, methyl jasmonate, and (R)-(-)-mellein, identified from the hairpencils of male Oriental fruit moths, attracts sex pheromone-releasing females several centimeters away. The chemicals thereby duplicate the behavioral effect elicited by hairpencil-displaying males during courtship; the chemicals also produce the herbal scent emanating from the hairpencils.

Reinvestigation of oak leaf roller sex pheromone components and the hypothesis that they vary with diet.

The sex pheromone of the oak leaf roller, Archips semiferanus, was identified as a specific blend (67:33) of trans-11- and cis-11-tetradecenyl acetates. The pheromone blend of females from a semisynthetic diet and from three oak species did not vary significantly. Males from these diets responsded in the laboratory and the field only to treatments approximating the 67 : 33 blend. These findings conflict with the hypothesis that the composition and perception of month sex pheromones vary with slight changes in diet.

Behavioral antagonism in the moth Helicoverpa zea in response to pheromone blends of three sympatric heliothine moth species is explained by one type of antennal neuron.

We have discovered a type of sensillum on the antennae of male corn earworm moths, Helicoverpa zea, that houses two types of receptor neuron (RN) that explains both the upwind flight attraction of males to their own species' pheromone blend and arrestment to the quite similar blends emitted by females of three other sympatric North American heliothine species. The first RN type is a large-spiking neuron that is most sensitive to (Z)-9-hexadecenal (Z9-16:Ald), the secondary H. zea pheromone component that along with the major component, (Z)-11-hexadecenal, causes attraction to the female. This RN is also responsive to (Z)-9-tetradecenal (Z9-14:Ald; not a H. zea pheromone component) at higher dosages. The sensitivity of this RN thus explains the attraction that has been observed in other studies when small proportions of Z9-14:Ald are added to Z11-16:Ald to mimic the conspecific blend. The second type of RN in this sensillum is a small-spiking neuron that is again responsive to Z9-14:Ald (which in larger proportions acts as a strong antagonist to upwind flight), but this RN is actually more sensitive to two other strong behavioral antagonists, (Z)-11-hexadecenyl acetate and (Z)-11-hexadecenol. Thus, activation of this single broadly tuned 'antagonist' RN could explain why H. zea males will orient only to their conspecific females. These three compounds are emitted by females of three other North American species, H. subflexa, H. phloxiphaga, and H. virescens, as agonists in their blends, which also contain the H. zea components Z11-16:Ald and Z9-16:Ald. This antagonist RN may also explain why a blend of Z11-16:Ald and a small amount of Z9-14:Ald is never as attractive to H. zea males as the conspecific blend. Enhanced specificity for the conspecific blend arises because the antagonist RN is never stimulated to fire, even when large proportions of the pheromone component, Z9-16:Ald, are added to Z11-16:Ald. When Z9-14:Ald is used instead of Z9-16:Ald, however, and the proportion of Z9-14:Ald becomes too great, the threshold of the antagonist neuron as well as that of the agonist neuron is exceeded, and the upwind flight response begins to be suppressed.

Effects of pheromone and plant volatile release rates and ratios on trapping Anoplophora glabripennis (Coleoptera: Cerambycidae) in China.

Native to China and Korea, the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), is a polyphagous wood-boring pest for which a trapping system would greatly benefit eradication and management programs in both the introduced and native ranges. Over two field seasons, a total of 160 flight intercept panel traps were deployed in Harbin, China, which trapped a total of 65 beetles. In 2012, traps using lures with a 1:1 ratio of the male-produced pheromone components (4-(n-heptyloxy)butanal and 4-(n-heptyloxy)butan-1-ol) designed to release at a rate of 1 or 4 milligram per day per component in conjunction with the plant volatiles (-)-linalool, trans-caryophyllene, and (Z)-3-hexen-1-ol caught significantly more A. glabripennis females than other pheromone release rates, other pheromone ratios, plant volatiles only, and no lure controls. Males were caught primarily in traps baited with plant volatiles only. In 2013, 10× higher release rates of these plant volatiles were tested, and linalool oxide was evaluated as a fourth plant volatile in combination with a 1:1 ratio of the male-produced pheromone components emitted at a rate of 2 milligram per day per component. Significantly more females were trapped using the pheromone with the 10-fold higher three or four plant volatile release rates compared with the plant volatiles only, low four plant volatile + pheromone, and control. Our findings show that the male-produced pheromone in combination with plant volatiles can be used to detect A. glabripennis. Results also indicate that emitters should be monitored during the field season, as release rates fluctuate with environmental conditions and can be strongly influenced by formulation additives.

Locating a compact odor source using a four-channel insect electroantennogram sensor.

Here we demonstrate the feasibility of using an array of live insects to detect concentrated packets of odor and infer the location of an odor source (∼15 m away) using a backward Lagrangian dispersion model based on the Langevin equation. Bayesian inference allows uncertainty to be quantified, which is useful for robotic planning. The electroantennogram (EAG) is the biopotential developed between the tissue at the tip of an insect antenna and its base, which is due to the massed response of the olfactory receptor neurons to an odor stimulus. The EAG signal can carry tens of bits per second of information with a rise time as short as 12 ms (K A Justice 2005 J. Neurophiol. 93 2233-9). Here, instrumentation including a GPS with a digital compass and an ultrasonic 2D anemometer has been integrated with an EAG odor detection scheme, allowing the location of an odor source to be estimated by collecting data at several downwind locations. Bayesian inference in conjunction with a Lagrangian dispersion model, taking into account detection errors, has been implemented resulting in an estimate of the odor source location within 0.2 m of the actual location.

Evaluating the use of male-produced pheromone components and plant volatiles in two trap designs to monitor Anoplophora glabripennis.

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), commonly known as the Asian longhorned beetle, is a wood-boring invasive species introduced from Asia to North America and Europe in solid wood packing material. Efficient monitoring traps are needed to assess population density and dispersal in the field and to detect new introductions at ports of entry. For this purpose, we conducted field trapping experiments in China in the summers of 2007 and 2008. In 2007, we tested Intercept panel traps hung on poplar trees. In 2008, we used Intercept panel traps hung on poplar trees, screen sleeve traps wrapped around poplar trunks, and Intercept panel traps hung on bamboo poles 20 m away from host trees. Traps were baited with A. glabripennis male-produced pheromone alone or in different combinations with plant volatiles. Traps baited with the male-produced pheromone alone caught significantly more females than control traps in both years. The addition of a mixture of (-)-linalool, (Z)-3-hexen-1-ol, linalool oxide, trans-caryophyllene, and trans-pinocarveol to the pheromone significantly increased trap catches of females, 85% of which were virgin. Screen sleeve traps baited with a combination of (-)-linalool and the pheromone caught the highest number of beetles overall in 2008, whereas traps placed on bamboo polls caught the lowest number. Although the logistics for the most effective implementation of a trapping program using a mixture of the pheromone and plant volatiles require additional studies, these results indicate that this pheromone has considerable promise as a monitoring tool for A. glabripennis in the field.

Attraction of Anoplophora glabripennis to male-produced pheromone and plant volatiles.

The male-produced pheromone of Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is an equal blend of 4-(n-heptyloxy)butan-1-ol and 4-(n-heptyloxy)butanal, was used in laboratory bioassays and in the greenhouse to determine its potential for attracting A. glabripennis adults. In modified "walking wind tunnels," virgin females were most attracted to the alcohol component, and virgin males were repelled by the pheromone blend at the lowest and highest amounts offered. Y-tube olfactometer bioassays also showed that females were significantly more attracted to the pheromone and its components than males were. However, males were more attracted to plant volatiles than females. Of 12 plant volatiles tested, delta-3-carene and (E)-caryophyllene were highly attractive to males, whereas (Z)-3-hexenyl acetate was repellent to males. Combining the male pheromone blend with (-)-linalool alone or with (Z)-3-hexen-1-ol attracted significantly more males than did the pheromone alone. We tested four trap designs in our quarantine greenhouse with eight different lures. The Intercept Panel traps and the hand-made screen sleeve traps caught more beetles than the Plum Curculio traps and Lindgren funnel traps. Intercept traps worked best when baited with male blend and (Z)-3-hexen-1-ol, whereas screen sleeve traps were most attractive when baited with (-)-linalool. Our findings provide evidence of the attractiveness of the A. glabripennis male-produced pheromone and suggest that it has a role in mate-finding. It is also a first step toward the development of an efficient trap design and lure combination to monitor A. glabripennis infestations in the field.

Odor detection in insects: volatile codes.

Insect olfactory systems present models to study interactions between animal genomes and the environment. They have evolved for fast processing of specific odorant blends and for general chemical monitoring. Here, we review molecular and physiological mechanisms in the context of the ecology of chemical signals. Different classes of olfactory receptor neurons (ORNs) detect volatile chemicals with various degrees of specialization. Their sensitivities are determined by an insect-specific family of receptor genes along with other accessory proteins. Whereas moth pheromones are detected by highly specialized neurons, many insects share sensitivities to chemical signals from microbial processes and plant secondary metabolism. We promote a more integrated research approach that links molecular physiology of receptor neurons to the ecology of odorants.

Real-time odor discrimination using a bioelectronic sensor array based on the insect electroantennogram.

Current trends in artificial nose research are strongly influenced by knowledge of biological olfactory systems. Insects have evolved over millions of years to detect and maneuver toward a food source or mate, or away from predators. The insect olfactory system is able to identify volatiles on a time scale that matches their ability to maneuver. Here, biological olfactory sense organs, insect antennae, have been exploited in a hybrid-device biosensor, demonstrating the ability to identify individual strands of odor in a plume passing over the sensor on a sub-second time scale. A portable system was designed to utilize the electrophysiological responses recorded from a sensor array composed of male or female antennae from four or eight different species of insects (a multi-channel electroantennogram, EAG). A computational analysis strategy that allows discrimination between odors in real time is described in detail. Following a training period, both semi-parametric and k-nearest neighbor (k-NN) classifiers with the ability to discard ambiguous responses are applied toward the classification of up to eight odors. EAG responses to individual strands in an odor plume are classified or discarded as ambiguous with a delay (sensor response to classification report) on the order of 1 s. The dependence of classification error rate on several parameters is described. Finally, the performance of the approach is compared to that of a minimal conditional risk classifier.

Inheritance of olfactory preferences II. Olfactory receptor neuron responses from Heliothis subflexa x Heliothis virescens hybrid male moths.

Single-cell electrophysiological recordings were obtained from olfactory receptor neurons (ORNs) in sensilla trichodea on male antennae of hybrids formed mainly by crossing female Heliothis subflexa with male Heliothis virescens ('SV hybrids'). We recorded from the A-, B-, and C-type sensilla trichodea, with the latter two types housing ORNs exhibiting response profiles to different pheromone components that we had previously found to be characteristic for each species. For both the B- and the C-type SV hybrid sensilla, most of the ORNs exhibited a spike amplitude and ORN co-compartmentalization within sensilla that more strongly resembled the ORNs of parental H. subflexa rather than those of H. virescens. The overall mean dose-response profiles of the ORNs in hybrid C- and B-type sensilla were intermediate between those of the H. virescens and H. subflexa parental type ORNs. However, not all hybrid ORNs were intermediate in their tuning spectra, but rather ranged from those that closely resembled H. subflexa or H. virescens parental types to those that were intermediate, even on the same antenna. The most noteworthy shift in ORN responsiveness in hybrid males was an overall increase in sensitivity to Z9-14:Ald exhibited by Z9-16:Ald-responsive ORNs. Heightened cross-responsiveness to Z9-14:Ald by hybrid ORNs correlates well with observed behavioral cross-responsiveness of hybrids in which Z9-14:Ald could substitute for Z9-16:Ald in the pheromone blend, a behavior not observed in parental types. The hybrid ORN shifts involving greater sensitivity to Z9- 14:Ald also correlate well with studies of hybrid male antennal lobe interneurons that exhibited a shift toward greater cross-responsiveness to Z9-14:Ald and Z9- 16:Ald. We propose that the differences between parental H. virescens, H. subflexa, and SV hybrid male pheromone ORN responsiveness to Z9-16:Ald and Z9-14:Ald are most logically explained by an increased or decreased co-expression of two different odorant receptors for each of these compounds on the same ORN.

Measured behavioural latency in response to sex-pheromone loss in the large silk moth Antheraea polyphemus.

Males of the giant silk moth Antheraea polyphemus Cramer (Lepidoptera: Saturniidae) were video-recorded in a sustained-flight wind tunnel in a constant plume of sex pheromone. The plume was experimentally truncated, and the moths, on losing pheromone stimulus, rapidly changed their behaviour from up-tunnel zig-zag flight to lateral casting flight. The latency of this change was in the range 300-500 ms. Video and computer analysis of flight tracks indicates that these moths effect this switch by increasing their course angle to the wind while decreasing their air speed. Combined with previous physiological and biochemical data concerning pheromone processing within this species, this behavioural study supports the argument that the temporal limit for this behavioural response latency is determined at the level of genetically coded kinetic processes located within the peripheral sensory hairs.

Behavioral responses of maleHeliothis virescens in a sustained-flight tunnel to combinations of seven compounds identified from female sex pheromone glands.

Each of the seven compounds that have been identified from femaleHeliothis virescens sex pheromone glands was examined for its ability to elicit sexual responses from male moths in a flight tunnel. The two compounds initially described as pheromone components, (itZ)-11-hexadecenal and (itZ)-9-tetradecenal, were necessary for behavioral activity to occur. Of the remaining five compounds, hexadecanal was most consistent in elevating behavioral activity of males when it was added to treatments. Live, calling females elicited greater sexual activity from males than did the 7-compound mixture on rubber septa.

Reproductive performance and longevity of female European corn borer, Ostrinia nubilalis: effects of multiple mating, delay in mating, and adult feeding.

In a recent study on the pheromone-mating disruption of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), we recorded a significant reduction in mating frequency, as well as a marked delay in mating in feral females captured in disruptant-treated fields. In order to be able to accurately interpret the results in terms of effective population control, the current study was undertaken on the effects of multiple matings and a delay in mating on reproductive performance. Female O. nubilalis that mated at least twice had significantly higher fecundity and fertility, compared with once-mated females. In addition, multiple-mated females deposited a significantly larger portion of their egg complement, relative to single-mated or unmated females. Females that experienced a 3-day delay in mating showed a significant reduction in fecundity compared with females that mated soon after emergence. A 1-week delay in mating resulted in a further reduction in fecundity and a near zero fertility. The effect of sugar feeding on reproduction was not significant. In general, unmated females lived longer than mated females, and sugar-fed mated females had a higher longevity than water-fed mated females.

Potential for evolution of resistance to pheromones : Worldwide and local variation in chemical communication system of pink bollworm moth,Pectinophora gossypiella.

FemalePectinophora gossypiella (Saunders) from most of the desert cotton-growing areas of southern California emitted significantly more pheromone in 1984 and 1985 than in preceding years (1982 and 1983). This increase amounted to almost 20% by 1985. It is unlikely that this small change would represent effective resistance to disruptant pheromones, but this increase could reflect the result of selection pressure imposed by the use of mating disruption for population control. A worldwide survey of emitted pheromone from this species found that there was much more variation in the emission rate than the blend ratio of the two pheromone components. The emitted blend ratio was remarkably consistent over time (in southern California) and throughout the worldwide range of the insect. Small differences in the blend ratio that were detected probably have no major biological significance because of the relatively broad response spectrum of males to changes in the blend of pheromonal components. Populations of males did not consist of several phenotypes, each with a different preference for specific blend ratios. Rather, the broad response spectrum to blend ratios in a population can be attributed to variation in the response of any individual. Therefore, selection for a response phenotype that is narrowly tuned to the blend emitted by females may be difficult.

Reiterative responses to single strands of odor promote sustained upwind flight and odor source location by moths.

We characterized single upwind surges of flying male Heliothis virescens moths in response to individual strands of pheromone generated experimentally in a wind tunnel. We then showed how this surge functions in this species as a basic 13.4-cm, 0.38-sec-long building block that is strung together repeatedly during typical male upwind flight in a normal pheromone plume. The template for a single iteration, complete with crosswind casting both before and after the straighter upwind surging portion, was exhibited by males flying upwind to pheromone and experiencing filament contacts just frequently enough to produce successful upwind flight to the source, as hypothesized by an earlier model. Also as predicted, with more frequent filament contact by males, only the straightest upwind portions of the surges were reiterated, producing direct upwind flight with little crosswind casting. Electroantennogram recordings made from males in free flight upwind in a normal point source pheromone plume further support the idea that a high frequency of filaments encountered under the usual pheromone plume conditions promotes only these repeated straight surges. In-flight electroantennogram recordings also showed that when filament contacts cease, the casting, counterturning program begins to be expressed after a latency period of 0.30 sec. Together these results provide a plausible explanation for how male and female moths, and maybe other insects, fly successfully upwind in an odor plume and locate the source of odor, using a surging-casting, phasic-tonic response to the onset and disappearance of each odor strand.