Identification of compounds produced by male hairpencil glands of corn earworm, Helicoverpa zea and their role in male autodetection and female mate acceptance

Heliothine moths represent some of the world's most important agricultural pest species. Helicoverpa zea (Corn Earworm) and Heliothis virescens (Tobacco Budworm) cause billions in damage and control costs worldwide each year. Given their economic importance, sex pheromones of many species have been studied for the development of management techniques such as trapping for population monitoring. The majority of pheromones identified and studied to date have been female‐produced sex pheromones. Less emphasis has been placed on male sex pheromones, associated with abdominal hairpencil structures that often function in courtship or to attract females. The present study examines the composition, detection and behavioural role of male H. zea hairpencil compounds in male autodetection and in courtship. Comparative examination of the hairpencil effluvia of H. zea and H. virescens did not reveal distinct differences in pheromone blend composition produced by males of these species. Electrophysiological testing demonstrated broad antennal neuron response in both males and females of H. zea to nine key stimuli, with sexual dimorphism present in each species. Behavioural assays suggested that odours released by male hairpencils are important in mate acceptance by female H. zea and may play a role in mate choice and species isolation. Wind tunnel observations indicated that key H. zea hairpencil odours (hexadecanyl acetate and octadecanyl acetate) also function in mate competition, antagonising responses of downwind conspecific males following a synthetic female sex pheromone plume. This finding provides insight regarding male–male detection and response to hairpencil compounds in H. zea.


INTRODUCTION
Heliothine moths belong to the subfamily of Heliothinae, with many species being distributed globally.The subfamily of Heliothinae includes about 25-28 genera and approximately 365 species (Cho et al., 2008;Hillier & Baker, 2016).The degree of host association between these different heliothine species is highly variable.Some species are highly host-specific while some exhibit wide polyphagy, wherein they use more than 100 plant species for feeding (Hillier & Baker, 2016).Most of heliothine species ($70%) are known to be oligophagous, feeding only on a few specific species of host plants, and the remaining 30% of species are known to be polyphagous, feeding on a vast number of host plant species (Cho et al., 2008).For example, larvae of Heliothis subflexa (Guenée, 1852) (Lepidoptera: Noctuidae) feed mainly on the fruits of Physalis spp.such as tomatillos, cape gooseberries and ground-cherries, whereas Schinia florida (Guenee, 1852) (Lepidoptera: Noctuidae) larvae feed exclusively on the buds of evening primrose (Onagraceae: Oenothera spp.) (Hardwick, 1965;Hillier & Baker, 2016).Species that belong to the genera Heliothis and Heliocoverpa are considered to be the most important agricultural pests (Cho et al., 2008).The Heliothis group includes some of the most important agricultural pests worldwide, causing massive crop losses each year, particularly in the developing world (Cho et al., 2008;Hillier & Baker, 2016).The estimated control costs per annum in the developing world are roughly about between US$ 3 billion and US$ 7 billion for the most prevalent agricultural pest species: H. armigera, H. zea, H. assulta, H. punctigera and H. virescens (Fitt, 1989;Hardwick, 1965;Hillier & Baker, 2016).Helicoverpa armigera, H. zea, Helicoverpa assulta (Guenée, 1852) (Lepidoptera: Noctuidae), Helicoverpa punctigera (Wallengren, 1860) (Lepidoptera: Noctuidae) and H. virescens are important pests on a range of food crops such as cotton, corn, sorghum, soybean, flax, tobacco and tomato (Hillier & Baker, 2016).Helicoverpa zea is a major polyphagous pest that has become one of the most constant insect pests of sweet corn, Zea mays (Burkness et al., 2009).Female moths of H. zea can lay over 2000 eggs within a 2-week period, ovipositing primarily on newly emerging silk tissues of corn.Once eggs hatch, emerging larvae then move down the silk to the ear tip, where they then start to feed on the corn kernels (Burkness et al., 2009).
Use of insect pheromones is one example of an alternate method for integrated pest management (Kirsch, 1988;Teal et al., 1986).Insect pheromones are used for multiple functions in integrated pest management.
The vast majority of pheromones are used as lures in traps for (1) monitoring populations to better time and determine the need for insecticidal applications, (2) direct control in mass trapping with pheromones, (3) attract and kill with pheromones or (4) mating disruption (Mishra et al., 2020;Teal et al., 1986;Tewari et al., 2014;Witzgall et al., 2010).
Understanding the biology and behaviour of heliothine moths is necessary for advancing novel techniques in integrated pest management.This will support development of crop production systems that optimize the use of natural resources while protecting the environment and increasing the output in a sustainable manner (Kirsch, 1988).
Males of many moths species have pheromone producing scent organs located on various body parts such as abdomen, thorax, legs and wings (Birch et al., 1989;Birch et al., 1990).The appearance of such organs can vary from simple scales and hair tufts to very complex eversible structures that have storage areas as well as perform intricate mechanisms such as exposure, expansion and concealment (Birch et al., 1990).The most complex scent-producing organs found in male Lepidoptera are called 'hairpencils' (Birch et al., 1990).In heliothine moths, hairpencil glands are located near the tip of the abdomen, associated with the valvae of the genitalia, and only exposed during elaborate courtship behaviour (Birch et al., 1990;Birch & Hefetz, 1987).The hairpencil glands of heliothine moths are made of hypertrophied trichogen cells, which are collected to form a brush.These moths have a pocket that they use to retract and conceal these hairpencil brushes when not in use but extend through the abdomen by muscle contraction and hydrostatic pressure during courting.In heliothines, hairpencil pheromone components are produced in the stobbel's glands within a glandular pocket near the abdominal tip.Pheromones soak the hairpencil brush while inverted and will then evaporate to the airstream when the hairpencils are everted (Birch et al., 1989;Birch et al., 1990).
Volatile chemicals produced by the hairpencil glands are important in courtship and have been demonstrated to influence conspecific female behaviour (Birch et al., 1989;Hillier & Vickers, 2004).
Electroantennography (EAG) performed on noctuid moths has shown that both male and females have the capability of detecting these volatile chemicals (Birch et al., 1990).In leek moths Acrolepiopsis assectella (Zeller ( 1839)) (Lepidoptera: Yponomeutoidea), male-produced pheromones can cause a female to become quiescent, acting as a female attractant, or an inhibitor for other approaching males (Lecomte et al. 1998).Male and female H. virescens respond electrophysiologically and behaviorally to components produced by male hairpencil pheromone glands (16-and 18-chain saturated alcohols and acetates), resulting in quiescence in activity in which females are subdued and competing males are antagonised from approaching another male during a courtship bout (Hillier & Vickers, 2004).
To date, the role of autodetection (perception of pheromones by the same sex and species as the emitter) has been sparsely documented in insects.The aim of this study is to examine the role of heliothine hairpencil compounds in female H. zea mating behaviour and to examine the function and potential influences of male-male autodetection on pheromone communication in heliothine moths.Our current hypothesis is that mate acceptance by female H. zea is species-specific and that odours released by male hairpencils are similarly important in mate acceptance by female H. zea and play a role in mate choice and species isolation.
Thus, we hypothesise that this is crucial to prevent mating mistakes by suitors of wrong species.We also hypothesise that key H. zea hairpencil components (16:OAc and 18:OAc) function to antagonize responses of conspecific competing males following a female sex pheromone plume.

Insects
Heliothis virescens and Helicoverpa zea used in this study were obtained from colonies maintained at the Acadia University, Nova Scotia, which were originally sourced from Frontier Agricultural Sciences (Newark, DE, USA), from colonies that have been in culture since 1998 without introduction of new genetic material.Larvae were reared on a premixed Tobacco Budworm diet until pupation.Pupae were sexed and maintained under a revised 14:10 dark-light cycle at 25 C and 60% relative humidity.Newly emerged adults were removed from rearing containers and stored in the same environmentally controlled insectary room.Small plastic cups filled with sugar water (one part sugar and four part water) ad libitum and a paper towel wick were provided for the emerging adult moths.All moths used for the experimentation were 2-7-day old virgins.
Female and male pheromone components were obtained from Bedoukian Research Inc (www.bedouklan.com).Stepwise volumetric decadic dilutions (10-0.01μg/μL) were made for each chemical in hexane and stored at À20 C.

Isolation and identification
A solid-phase micro-extraction (SPME) fibre (diameter of 100 μm and needle size 24 ga) coated with polydimethylsiloxane (SigmaAldrich/ MilliporeSigma, Oakville, ON, Canada) was used to extract hairpencil compounds using methods adapted from Hughes and Cardé (2020).SPME collections were made in the last hour of photophase and the first 5 h of scotophase to make sure the pheromone gland had not been emptied prior to the extraction.The abdomen of male moths was pressed gently to expose the hairpencil.The SPME fibre was then rubbed along the exposed hairpencil gland for 2 min.The SPME fibre with collected volatiles was injected into a Varian 450 GC under a spitless mode of 1:20 for 1 min and was run for 35 min.The GC was equipped with a Rxi ® -5silms non-polar capillary column (30 m Â 0.25 mm, film thickness 0.25 mm; Restek Corporation, State college, PA, USA).Temperatures of the injector and FID(Flame Ionization Detector) were 250 and 320 C, respectively, with helium as carrier gas at a flow rate of 1.0 mL/min.The starting temperature of the oven was 50 C, which then increased by 10 C/min for 25 min.
The temperature was held at 300 C for 7.5 min.Retention times of peaks of hairpencil compounds collected by the SPME fibre were compared with pheromone standards for identification.Peak areas of the compound identified in hairpencil extraction and standard compounds were then used to calculate the amounts of those compounds present in the hairpencil extraction.
Peak retention times and peak areas from pheromone gland extracts were compared to standard curves established from known standards to identify components and amounts present.Five standard runs were completed at concentrations between 10 ng and 100 μg for all standard compounds.Peak areas of standard curves were measured, and a line of best fit was fitted through the mean peak areas at each concentration.The equation of the fitted line was used to calculate concentration of gland extract components.Each gland component concentration was calculated using the standard curve equation corresponding to the identified compound (i.e., concentration of peaks identified as 18:OAc were calculated using the equation for 18:OAc standards).Kruskal-Wallis one-way analysis of variance and Dunn's multiple comparison test were used to determine the statistical significance ( p < 0.05) of hairpencil concentrations between each heliothine moth species.

Electrophysiology
EAG was used to measure the relative sensitivity of male and female H. zea to various components of male hairpencil glands and known components of H. zea female sex pheromone, as a means to screen for those compounds with greater behavioural relevance.Furthermore, by comparing male and female responses, it could help with determining if certain compounds might be more important as cues for the same or opposite sexes.Stimuli were prepared from previously diluted chemicals at four stimulus loads: 100 ng, 1 μg, 10 μg and 100 μg.This was done by soaking 1 cm Â 3 cm rectangle sections of filter paper with 10 μL of prepared odorant aliquots.The filter papers were then placed into 14.6 cm glass pipette.A 1000 μL micropipette tip was used to cover the wide end of the glass pipette.Another 1000 μL pipette tip blocked with dental wax was placed on the top of that pipette tip.The prepared odorant stimulus cartridges were then wrapped in aluminium foil and stored at À20 C until use.
EAG was prepared by decapitating virgin H. zea moth and attaching the head to the ground electrode of a EAG probe (Syntech, Hilversum, The Netherlands).Each side of the EAG probe was coated with electrode gel when mounting the antennae and head to improve the conductivity of signals and to prevent desiccation of the head and antennae.The preparation was mounted on the EAG headstage and micromanipulated into a humidified airstream.A constant flow of charcoal-filtered, humidified air was blown through a glass air transfer line at a rate of 0.5 L/min over the antennae on the probe.A section of plastic tubing that has a constant air flow, which is further connected to the air transfer line, was used to connect the stimulus cartridges.Odorants in the stimulus cartridges were introduced to the stream of air that flows over the antennae at random order, at increasing concentrations by pushing a stimulus pedal attached to a CS-55 stimulus controller (Syntech, Hilversum, The Netherlands).This allowed humidified air to flow through the glass transfer line to the air transfer line.Each stimulus was tested in random order, at ascending concentrations, with 1 min interstimulus interval between each concentration and between different odorants.Hexane was tested as a control at the beginning and the end of each experimental run.An Intelligent Data Acquisition Controller-4 (IDAC-4 ® , Syntech, Hilversum, The Netherlands) was used to collect the EAG signals.
Electroantennogram peaks were measured for amplitude and standardised by transforming EAG amplitudes into a ratio relative to the response of hexane controls.Mean hexane amplitude was calculated for each experimental run.To calculate odorant ratios, experimental odorant peak amplitudes were divided by the hexane mean of their respective run.Mean relative ratios were then calculated for each odorant at all tested stimulus loads.Data were transformed using square root transformation to fit into normal distribution.

Behavioural assay
Behavioural assays were conducted in a chamber made out of Plexiglas ® circles.The characteristics of the behavioural chamber have been described previously (Hillier & Vickers, 2004).A dark room illuminated with a single red incandescent light bulb and a temperature ranged from 20.6 to 22.0 C was used to conduct the behavioural experiments.The top and bottom of this chamber were made out of Plexiglas ® circles with 2 mm thickness and 17.5 cm diameter, with walls of mesh screening (3 mm size and 6 cm height).An epoxy was used to glue the mesh screening to Plexiglas ® circles, and the stability of the chamber was ensured by sticking three plastic support pillars to the inside of the walls.The top Plexiglas ® plate was drilled to make three holes in which the tips of each support pillar would fit (making top removable) and the rotation of the chamber in a hole on a wooden base was ensured by placing a screw in the middle of the bottom plate.The chamber was then placed in front of a vacuum line (a constant airflow at a rate of 1.0 m/s).Rotation of the mating chamber on its base was necessary to ensure that (1) the female moths were upwind from males before the start of courtship and (2) both moths were upwind during video recording.All mating trails were recorded by using a Sony ® Handycam DCR-SR45 digital video camera recorder, and once recorded, they were stored on an external memory source.

Hairpencil ablation assays
Hairpencil ablation trials were conducted to determine if odours present in male H. zea hairpencils played a role in female H. zea mate acceptance.Odour replacement trials were conducted on pairs of adults, virgin H. zea moths, by pairing females with conspecific males, which have been hairpencil ablated, and synthetic hairpencil blends added mimicking conspecific and heterospecific males.Two different experiments were conducted: (i) normal males (without hairpencil ablation) with normal females and (ii) hairpencil-ablated males plus an odour source of either male H. virescens or H. zea hairpencil extract (1 ME) with females; and hairpencil-ablated males plus a control odour source (hexane) with females.All ablation surgeries were conducted before scotophase (reversed light cycle) on the day of experimentation, and moths were returned to the environmental chamber until 1 h before the experiment (when they were removed for acclimation to room conditions).Male hairpencil surgery was conducted to remove as much of the hairpencils as possible by gently squeezing the abdomen to expose the hairpencils that were then trimmed.
Hairpencil odours of male H. zea and H. virescens were extracted by cutting the tip of their abdomen and immersing in a 2 mL glass vial containing 100 μL of hexane such that one pair of hairpencils (1 ME) = 100 μL hexane (10 hairpencils/1000 μL hexane).The tip of the abdomen was soaked in the hexane for a maximum of 1 min and was removed afterwards.Hairpencil extract (1 ME) of 100 μL was reduced by concentration to a 10-20 μL under N 2 , which was then loaded on a 1 cm diameter filter paper disc (Whatman No.4) prior to behavioural assays.The filter paper was left in the fume hood, allowing hexane to evaporate.
An odour source (1 cm diameter filter paper) attached to an alligator clip with 1 ME of H. zea or H. virescens hairpencil extract was introduced upwind from the female H. zea moth while ablated moth was courting.Hundred females were tested for their behavioural responses to each hairpencil extracts.Control trials (n = 100) were conducted using 10 μL of hexane applied to filter paper.'Normal' behavioural trials (n = 190) were conducted between normal male and female H. zea.The wire post and alligator clip were rinsed with acetone between the trials, to prevent crosscontamination of odour sources.In all courtship trials, female moths were first placed in the assay chamber and observed for calling behaviour.Only females that were consistently 'calling' (stationary, with wings fanning and ovipositor exposed) were used in the experiments.Generally, calling females remained on the chamber's walls and were rotated to the most upwind position, ventrally orientated to the video camera.The cage's downwind portion was then filled with single males (all ablation experiments were performed only on single pairs of males and females).Behaviour of moths was observed for 5 min.If no interaction was seen after 5 min (e.g., male unable to identify female), the trial was ended by removing both moths.If courtship was started during the first 5 min of observation, the trial was extended for another 5 min from the start of courtship.Female moth behaviours, such as fly away, move away, antennal flicking, wing fanning, ovipositor extension, abdominal extension, dragging as well as the number of mating attempts and success/failure to copulate (as evidenced by successful clasping of genitalia), were recorded in response to specific male courtship attempts.Frequency of moving away, flying away, antennal flicking, wing fanning, ovipositor extension, abdominal extension, dragging, copulation attempts and successful clasping was compared between separate treatments of conspecific and heterospecific mating pairs.A chi-square (χ 2 ) 2 Â 2 test of independence with Yates' correction was used to determine significant differences in behavioural frequency between inter-specific hairpencil extracts, hexane controls and normal male and female pairs.

Wind tunnel assay
Wind tunnel assays were performed to determine male-male autodetection by investigating the effects of hairpencil odorants on male flight responses to female sex pheromones.Virgin male H. zea moths of 3-7 days old were used for the experiment.A wind tunnel (2.5 Â 1.14 Â 1.14 m; L Â H Â W) illuminated with red LED light was used.Wind speed (0.47-0.60 m/s), temperature (21.1-22.0C) and relative humidity (25.4%-45.5%)were maintained.A wire cage (3 cm diameter Â 5 cm high) within a plastic container was used to place individual male insects before scotophase on the day of experimentation.During the second hour of scotophase, containers were returned to an environmental chamber.Individual wire cages containing moths were then moved to the wind tunnel room, allowing them to acclimatise to the room conditions.Odour blends were then introduced to the wind tunnel by attaching a rubber septa loaded with an odour blend to an alligator clip on a metal rod.The distance between the odour source and the moth 'take-off' platform was 1.5 m, and the odour source was 24 cm above the wind tunnel floor.The takeoff platform was set 50 cm upwind of the exhaust vent, in the centre to intersect the pheromone plume.
Pheromone inoculated rubber septa were prepared before beginning the wind tunnel experiments.Septa were first cleaned and stretched before using.Septa were put in a 250 mL bottle with a cover and poured enough dichloromethane (DCM) to cover.The bottle was left in the fume hood for 2 h, allowing septa to soak and dry.After 2 h, excess DCM was drained.Septa were spread out on clean glass Petri dishes for 24 h in the fume hood and later put in a storage bottle for further use.Cleaned septa were then used to load with pheromone components.Two main hairpencil compounds produced by male H. zea specifically; 18:OAc and 16:OAc were used to test the flight responses of male H. zea moths.Rubber septa soaked with 16:OAc and 18:OAc following a female sex pheromone plume (Z11-16:Ald and Z9-16:Ald in 95:5) were used for the assays.Septa that loaded with the synthetic female sex pheromone blend were used as controls.16:OAc and 18:OAc were tested in three concentrations: 1, 10 and 100 μg.A mixture of 95 μg Z11-16:Ald + 5 μg Z9-16:Ald ('H.zea 2-mix', a known attract for male H. zea (Hillier & Vickers, 2007)) was loaded into the septa.16:OAc and 18:OAc were then admixed to clean rubber septa at increasing dosages: 1, 10 and 100 μg (or 1%, 10% and 100% relative to the concentration of Z11-16:Ald in the mixture), which were then used to test the effects of hairpencil odorants in wind tunnel.Septa admixed with pheromone mixtures were then filled with 150 μL of DCM at the end and left in the fume hood for 24 h before testing.
A group of 10-20 insects were used to test a single odourconcentration treatment each day, along with 10-20 insects flown to the control pheromone mixture.For each trial, moths were released by inverting their cage and placing in on the 'take-off' platform to permit the moth to exit.Behavioural responses observed and quantified were (Hillier & Vickers, 2007): • take flight: moth 'activates' begins wing fanning and engages in flight.
• upwind flight: moth locates plume and begins characteristic counterturning flight towards odour source.
• 75 cm: moth continues upwind counterturning flight beyond half the distance between the take-off platform and the odour source.
• 5 cm: moth continues upwind counterturning flight to within 5 cm of the odour source A chi-square (χ 2 ) 2 Â 2 test of independence with Yates' correction was used to compare the percentage of males engaging in each behavioural step in wind tunnel assays.Results were considered statistically significant if p < 0.05.All statistical analysis were done using the R software of i386 4.0.3version.

Hairpencil compound identification
GC-FID(Gas Chromatography Flame Ionization Detector) analysis was conducted on gland extracts of male H. zea (n = 20) and H. virescens (n = 20).Compound peaks were clearly separated and identifiable based on retention times.Four compounds (18:OAc, 16:OAc, 18:OH and 16:OH) were identified within male hairpencil glands of both H. zea and H. virescens using analytical standards.All standards were detected within any single gland in different concentrations.In both species, 16:OAc and 18:OH recorded the highest concentration (Table 1).Both species had 16:OH in least concentration (Table 1).
Notably, amounts of each compound extracted from effluvia were very similar between species.

Electroantennography
Male and female H. zea were tested for EAG responses to nine compounds.Twenty individuals per sex were used for the analysis.
All individuals demonstrated neuronal responses to all nine compounds at all stimulus loads tested (Figure 1).There were significant interactions between sex and stimulus in EAG responses ( p < 0.05, F value = 2.342, df = 8).However, there were no significant interactions between stimulus and concentration ( p = 0.53, The EAG responses of males to 16:OAc, Z11-16:OH, Z11-16:Ald and Z9-14:Ald gradually increased with increasing concentration.
However, females did not show any concentration-dependent increase in sensitivity to any stimuli except increased sensitivity to 16: OAc, 16:OH and 18:OAc at 1 μg (Figure 1).The response then gradually decreased at 10 and 100 μg.Males had stronger responses at all stimulus loads tested, but the significant differences were seen only in
When exposed to H. virescens hairpencil odour, female H. zea moved away more frequently, though this was not significantly different from exposure to H. zea hairpencil odour (14%; Figure 2).There was no significant difference in antennal flicking when exposed to H. zea and H. virescens hairpencil odours, but a significant difference was observed when females were paired with normal males (59%) and hairpencil-ablated males exposed to H. zea (36%), H. virescens hairpencil odour (32%) or hexane (30%).

Wind tunnel assay
There was an overall reduction in activation, and more frequent prematurely ceased upwind flight and contact to the odour source in male H. zea in response to 16:OAc and 18:OAc added to a synthetic female pheromone attractant mixture (95:5 Z11-16:Ald and Z9-16: Ald).This effect was concentration-dependent for both hairpencil components tested (Figure 3).Upwind flight and contact with the odour source decreased gradually in the behavioural sequence and in response to increasing concentration of 18:OAc.The lowest frequency of source contact was recorded at 100 μg of 18:OAc (4.5%) compared with 10 μg (15%) and 1 μg (25%).The frequency of moth continuing upwind counterturning flight beyond the half the distance between the take-off platform and the odour source (75 cm) was significantly different between positive control and 10 μg of 18:OAc (p < 0.01, χ 2 = 6.827, df = 1).Even though moths had a lower frequency of contacting 10 μg 18:OAc (15%) applied odour source compared with positive control (45%), the difference was not significant

DISCUSSION
Hairpencil compositions of identified compounds in this study were not different between H. zea and H. virescens; however, differential behavioural responses by female H. zea suggest the presence of other unidentified compounds that may permit females to actively distinguish between hairpencil extracts from closely related sympatric species (Hillier & Vickers, 2004).Males of both species produce and detect (and females detect) 16:OAc, 18:OAc and their alcohol counterparts; however, given their similarity, these compounds cannot function unilaterally to provide for species isolation, and ergo may function in concert with other cues (possibly additional hairpencil components, differential production or release rates, or other nonvolatile cues).An unanticipated finding was that concentrations of various hairpencil compounds detected in effluvia were virtually identical in this study.Given previous studies that have documented variable gland and effluvial compositions (Hillier & Vickers, 2004;Teal & Tumlinson, 1989), this seems an anomalous outcome, which may reflect limitations in our effluvial collection.The current study focused on the use of SPME-collected effluvia; however, it is possible that other extraction methods may provide more discrete differences in quantification and identification of compounds.Finally, recent work by Liu et al. (2023) has also shown that in H. virescens, male hairpencils release a mosaic of compounds, including plant derivatives, which may also influence conspecific courtship behaviour.
Hexadecenyl acetate and 18:OAc play a key role in behavioural responses of conspecific males.Both compounds act as antiaphrodisiacs to competing conspecific males, inhibiting the upwind flight and orientation to a source of synthetic female sex pheromone.
We hypothesise that both male and female H. zea respond to 16:OAc and 18:OAc in a similar way as has been documented in H. virescens; with both males and females responding to these hairpencil compounds by eliciting quiescent behaviour (Hillier & Vickers, 2004).
Overall, EAG sensitivity to pheromone components was relatively similar across all compounds tested.Dose-response to increasing concentrations of stimuli was not robust, perhaps indicating that a broader range of concentrations warranted testing to observe sigmoid responses curves.It is possible that the lowest dose of various stimuli is already near response maxima for both sexes.Primary conclusions that can be made are that both sexes exhibited some response to each of compounds tested and that males were consistently more sensitive.Interestingly, all males responded most strongly to (Z)9-14: Ald, a component found in the pheromone blend of H. virescens.Male H. zea also had relatively strong EAG responses to 18:OAc and 16: OAc (compounds with known pheromone function in H. subflexa and H. virescens (Hillier & Vickers, 2004;Hillier & Vickers, 2011).Both compounds suppressed upwind flight of male H. zea to synthetic sex pheromone and may act as an anti-aphrodisiac during courtship, inhibiting the approach of competing conspecific males.
Female behavioural responses to conspecific and inter-specific hairpencil extracts showed that females can distinguish between hairpencil extracts when presented in concert with a courting male.In particular, this study provides evidence that hairpencil odours may act to: (i) produce quiescence or make females susceptible to mating and (ii) identify appropriate suitors (conspecifics) from other species.This suggests that H. zea hairpencil odour has an arrestment (prevent females from moving away) and aphrodisiac (make female more receptive to mating) effect, similar to other observations from other heliothines (Hillier & Vickers, 2004;Hillier & Vickers, 2011).
The precision of the mixture of compounds in male hairpencils is likely important for mate acceptance by females and in reproductive isolation (by escaping from incorrectly 'smelling' males).Four It is important to note that while lures were loaded at similar rates, the relative molecular weights and vapour pressures will have an effect of the emission rates of the pheromone mixtures (Hillier & Vickers, 2007).Hexadecenyl acetate has a higher molecular weight compared with 18:OAc, thus the emission rate of 16: OAc will be higher than 18:OAc since it has a higher vapour pressure compared with 18:OAc.However, because the concentration of 16:OAc is substantially higher in male H. zea hairpencil gland extracts (the natural concentration of each in airborne effluvia is unknown (Teal & Tumlinson, 1989)), it is uncertain whether 18: OAc or 16:OAc is more important in mediating male-male interaction (notwithstanding that natural composition of hairpencil effluvia is a mixture containing both compounds).
Overall, this study demonstrates that male hairpencil extracts cause quiescence in female H. zea, and selected components inhibit upwind flight of males towards a calling female, as has been reported in H. virescens (Hillier & Vickers, 2004;Hillier & Vickers, 2007).Future research is required for behavioural testing of blends of these key compounds, as well as with natural hairpencil extracts to determine if mixtures or unidentified compounds are important male-female or male-male communication.This study provides an insight on antennal sensitivity of male and female H. zea and the effect of hairpencil odours on male and female behavioural responses, as well as new insights regarding putative pheromones used in courtship by this moth species.
Three-way analysis of variance and adjusted Tukey comparison test were run in R (R Foundation for Statistical Computing, 2021) to determine the statistical significance of antennal responses for each odour compound between sexes.
U R E 1 Comparison of mean ratio of EAG response amplitude ± SE relative to hexane controls of males (n = 20) and females (n = 20) of Helicoverpa zea for each compound tested at four different concentrations (0.1, 1, 10 and 100 μg).
(p = 0.085, χ 2 = 2.976, df = 1).The frequency of upwind flight and flying towards the odour source further decreased in response to 100 μg of 18:OAc compared with positive control.The differences of all flight responses between 18:OAc (100 μg) mixture and positive control were significant except for taking flight.Behavioural trials with 16:OAc added to an attractive 2-component pheromone blend also showed similar results.The frequency of upwind flight, continuous upwind counterturning flight and locating odour source gradually decreased in response to 16:OAc compared with positive control.This decrease was concentration-dependent.The differences in flight responses Occurrence of female Helicoverpa zea behavioural responses to a courting hairpencil-ablated male, during exposure to filter papers loaded with one male equivalent of conspecific: H. zea and inter-specific (Heliothis virescens) hairpencil extracts (n = 100); and a control of a normal pair of male and female H. zea (n = 100).Control trials were conducted using hexane (n = 100).Bars with no letters in common are significantly different from one another for a specific behaviour (p < 0.05).between 100 μg of 16:OAc and positive control were significantly different in all responses except for take flight (upwind flight, p = <0.0001;75 cm, p = <0.0001; 5 cm, p = <0.0001and source p < 0.0001).
hairpencil compounds were discovered to be significant in triggering female acceptance posture in studies on A. assectella Zeller (Lepidoptera: Acrolepiidae) (Thibout et al 1994); however, the blends tested did not seem to indicate any synergy between the most active compounds.It is unclear whether hairpencil compounds act in a similar manner on H. zea females, and this requires additional investigation.Wind tunnel experiments showed that the addition of 1%-100% of 16:OAc or 18:OAc relative to an attractive pheromone mixture antagonises upwind flight of male H. zea.Hillier and Vickers (2007) reported an similar, concentration-dependent antagonistic effect on the upwind flight of male H. virescens caused by addition of 16:OAc and 18:OAc to an attractive synthetic pheromone blend.Because upwind flight to synthetic female sex pheromone is suppressed by coincident presentation of 16:OAc and 18:OAc, H. zea male hairpencil odours may act as an antiaphrodisiac during courtship, repelling competing conspecific males.