Dancing in the purple rain: color affinity and oviposition choices in Aedes sierrensis (Diptera: Culicidae).

The western tree hole mosquito, Aedes sierrensis (Ludlow) (Diptera: Clucidae), is a pestiferous mosquito with a range extending over the entire pacific seaboard and into portions of the intermountain west. As a peridomestic heartworm vector, it demands at least some level of surveillance to understand its abundance. However, the species is refractory to a majority of conventional vector surveillance approaches for tracking mosquitoes. To find more options for Aedes sierrensis surveillance, a variety of oviposition attractants were evaluated in arena-style choice assays using colony reared adults. A range of infusion treatments (e.g., alfalfa, oak, and beetroot) were examined and then combined with investigations of liquid color as well as ovicup color and entryway position. These studies revealed that Ae. sierrensis have an affinity for purple coloration, plain water, and larger entryway sizes for oviposition cups. A prototype ovicup was 3D-printed using purple filament and multiple types of entryways, and used to re-test infusion waters. No particular attraction differences were detected after normalizing for purple color. Comparisons to black 3D-printed cups yielded surprising observations that male mosquitoes also aggregated on purple cups while females sheltered, but not necessarily oviposited, in black cups. Although this was only a laboratory-based assessment, these studies provide useful information for future field trials of potential oviposition traps for surveillance of Ae. sierrensis.

Moth pheromone-selective projection neurons with cell bodies in the antennal lobe lateral cluster exhibit diverse morphological and neurophysiological characteristics.

Olfactory projection neurons convey information from the insect antennal lobe (AL) to higher brain centers. Previous reports have demonstrated that pheromone-responsive projection neurons with cell bodies in the moth medial cell cluster (mcPNs) predominantly have dendritic arborizations in the sexually dimorphic macroglomerular complex (MGC) and send an axon from the AL to the calyces of the mushroom body (CA) as well as the lateral horn (LH) of the protocerebrum via the medial AL tract. These neurons typically exhibit a narrow odor tuning range related to the restriction of their dendritic arbors within a single glomerulus (uniglomerular). In this study, we report on the diverse physiological and morphological properties of a group of pheromone-responsive olfactory projection neurons with cell bodies in the AL lateral cell cluster (MGC lcPNs) of two closely related moth species. All pheromone-responsive lcPNs appeared to exhibit "basket-like" dendritic arborizations in two MGC compartments and made connections with various protocerebral targets including ventrolateral and superior neuropils via projections primarily through the lateral AL tract and to a lesser extent the mediolateral antennal lobe tract. Physiological characterization of MGC lcPNs also revealed a diversity of response profiles including those either enhanced by or reliant upon presentation of a pheromone blend. These responses manifested themselves as higher maximum firing rates and/or improved temporal resolution of pulsatile stimuli. MGC lcPNs therefore participate in conveying diverse olfactory information relating to qualitative and temporal facets of the pheromone stimulus to a more expansive number of protocerebral targets than their mcPN counterparts.

Animal Communication: When I'm Calling You, Will You Answer Too?

Male moths compete to arrive first at a female releasing pheromone. A new study reveals that additional pheromone cues released only by younger females may prompt males to avoid them in favor of older but more fecund females.

Transplant Antennae and Host Brain Interact to Shape Odor Perceptual Space in Male Moths.

Behavioral responses to odors rely first upon their accurate detection by peripheral sensory organs followed by subsequent processing within the brain's olfactory system and higher centers. These processes allow the animal to form a unified impression of the odor environment and recognize combinations of odorants as single entities. To investigate how interactions between peripheral and central olfactory pathways shape odor perception, we transplanted antennal imaginal discs between larval males of two species of moth Heliothis virescens and Heliothis subflexa that utilize distinct pheromone blends. During metamorphic development olfactory receptor neurons originating from transplanted discs formed connections with host brain neurons within olfactory glomeruli of the adult antennal lobe. The normal antennal receptor repertoire exhibited by males of each species reflects the differences in the pheromone blends that these species employ. Behavioral assays of adult transplant males revealed high response levels to two odor blends that were dissimilar from those that attract normal males of either species. Neurophysiological analyses of peripheral receptor neurons and central olfactory neurons revealed that these behavioral responses were a result of: 1. the specificity of H. virescens donor olfactory receptor neurons for odorants unique to the donor pheromone blend and, 2. central odor recognition by the H. subflexa host brain, which typically requires peripheral receptor input across 3 distinct odor channels in order to elicit behavioral responses.

Male moths optimally balance take-off thoracic temperature and warm-up duration to reach a pheromone source quickly.

Animal activities, such as foraging and reproduction, are constrained by decisions about how to allocate energy and time efficiently. Overall, male moths invest less in reproduction than females, but they are thought to engage in a scramble competition for access to females that advertise readiness to mate by releasing sexual pheromones. However, before male moths can follow the pheromone, they often need to heat their flight muscles by shivering to produce sufficient power for sustained flight. Here, we show that Helicoverpa zea males that sense the female pheromone at high ambient temperatures take off with higher thoracic temperature, shiver for less time and warm up faster than males tested at lower ambient temperatures. These higher take-off temperatures translate into higher airspeeds, underscoring the importance of thoracic temperature for flight performance. Furthermore, shorter combined duration for warm-up and pheromone-mediated optomotor anemotaxis is consistent with the idea that males engage in scramble competition for access to females in nature. Our results strongly suggest that male moths minimize the time between perceiving the female's pheromone signal and arriving at the source by optimizing thermoregulatory behaviour and temperature-dependent flight performance in accordance with ambient temperature conditions. Our finding that moths engage in a trade-off between rapid flight initiation and suboptimal flight performance suggests a sensorimotor control mechanism that involves a complex interaction with the thermal environment.

Female pheromones modulate flight muscle activation patterns during preflight warm-up.

At low ambient temperature Helicoverpa zea male moths engage in warm-up behavior prior to taking flight in response to an attractive female pheromone blend. Male H. zea warm up at a faster rate when sensing the attractive pheromone blend compared with unattractive blends or blank controls (Crespo et al. 2012), but the mechanisms involved in this olfactory modulation of the heating rate during preflight warm-up are unknown. Here, we test three possible mechanisms for increasing heat production: 1) increased rate of muscle contraction; 2) reduction in mechanical movement by increased overlap in activation of the antagonistic flight muscles; and 3) increased activation of motor units. To test which mechanisms play a role, we simultaneously recorded electrical activation patterns of the main flight muscles (dorsolongitudinal and dorsoventral muscles), wing movement, and thoracic temperature in moths exposed to both the attractive pheromone blend and a blank control. Results indicate that the main mechanism responsible for the observed increase in thoracic heating rate with pheromone stimulation is the differential activation of motor units during each muscle contraction cycle in both antagonistic flight muscles. This additional activation lengthens the contracted state within each cycle and thus accounts for the greater heat production. Interestingly, the rate of activation (frequency of contraction cycles) of motor units, which is temperature dependent, did not vary between treatments. This result suggests that the activation rate is determined by a temperature-dependent oscillator, which is not affected by the olfactory stimulus, but activation of motor units is modulated during each cycle.

Pheromone mediated modulation of pre-flight warm-up behavior in male moths.

An essential part of sexual reproduction typically involves the identification of an appropriate mating partner. Males of many moth species utilize the scent of sex pheromones to track and locate conspecific females. However, before males engage in flight, warm-up by shivering of the major flight muscles is necessary to reach a thoracic temperature suitable to sustain flight. Here we show that Helicoverpa zea males exposed to an attractive pheromone blend (and in some instances to the primary pheromone component alone) started shivering earlier and took off at a lower thoracic temperature than moths subjected to other incomplete or unattractive blends. This resulted in less time spent shivering and faster heating rates. Two interesting results emerge from these experiments. First, the rate of heat generation can be modulated by different olfactory cues. Second, males detecting the pheromone blend take off at lower thoracic temperatures than males exposed to other stimuli. The take-off temperature of these males was below that for optimal power production in the flight muscles, thus generating a trade-off between rapid departure and suboptimal flight performance. Our results shed light on thermoregulatory behaviour of unrestrained moths associated with the scramble competition for access to females and suggest ecological trade-offs between rapid flight initiation and sub-optimal flight performance.

Antennal lobe organization in the slender pigeon louse, Columbicola columbae (Phthiraptera: Ischnocera).

This study reports on the structure of the antennal lobe of the pigeon louse, Columbicola columbae. Anterograde staining of antennal receptor neurons revealed an antennal lobe with a few diffuse compartments, an organization distinct from the typical spheroidal glomerular structure found in the olfactory bulb of vertebrates and the antennal lobe of many other insects. This anatomical arrangement of neuronal input is somewhat reminiscent of the aglomerular antennal lobe previously reported in psyllids and aphids. As in psyllids, reports on the odor-mediated behavior of C. columbae suggest that the olfactory sense is important in these animals and indicates that a glomerular organization of the antennal lobe may not be necessary to subtend odor-mediated behaviors in all insects. The diffuse or aglomerular antennal lobe organization found in these two Paraneopteran insect orders might represent an independently evolved reduction due to similar ecological constraints.

Hairpencil volatiles influence interspecific courtship and mating between two related moth species.

Reproductive isolation between sympatric, closely related species can be accomplished through a variety of pre-zygotic isolating mechanisms, including courtship-signaling behavior that involves pheromones. In the moths Heliothis virescens and H. subflexa, males display abdominal hairpencils (HP), which release volatile chemicals during courtship. In this study, we demonstrated that HP volatiles released by male H. subflexa function to improve mating success with conspecific females. Interspecific mating experiments were conducted to determine any influence of HP volatiles on species isolation. Female H. virescens and H. subflexa were observed during courtship with males of the other species, following either sham-operation or ablation of HPs, both with and without concurrent presentation of HP volatiles. Mating success was improved by co-presentation of HP extract from males of the same species during courtship. Ablation of HPs improved mating between H. subflexa females and H. virescens males. During interspecific matings, male H. virescens attempted copulation less frequently in the presence of H. virescens HP extract, though H. subflexa males were not affected by the presence of H. subflexa HP extract. This suggests that HP volatiles produced by males of each species may inhibit mating between species through effects on males (H. virescens) and females (H. subflexa).

Sexual isolation of male moths explained by a single pheromone response QTL containing four receptor genes.

Long distance sexual communication in moths has fascinated biologists because of the complex, precise female pheromone signals and the extreme sensitivity of males to specific pheromone molecules. Progress has been made in identifying some genes involved in female pheromone production and in male response. However, we have lacked information on the genetic changes involved in evolutionary diversification of these mate-finding mechanisms that is critical to understanding speciation in moths and other taxa. We used a combined quantitative trait locus (QTL) and candidate gene approach to determine the genetic architecture of sexual isolation in males of two congeneric moths, Heliothis subflexa and Heliothis virescens. We report behavioral and neurophysiological evidence that differential male responses to three female-produced chemicals (Z9-14:Ald, Z9-16:Ald, Z11-16:OAc) that maintain sexual isolation of these species are all controlled by a single QTL containing at least four odorant receptor genes. It is not surprising that pheromone receptor differences could control H. subflexa and H. virescens responses to Z9-16:Ald and Z9-14:Ald, respectively. However, central rather than peripheral level control over the positive and negative responses of H. subflexa and H. virescens to Z11-16:OAc had been expected. Tight linkage of these receptor genes indicates that mutations altering male response to complex blends could be maintained in linkage disequilibrium and could affect the speciation process. Other candidate genes such as those coding for pheromone binding proteins did not map to this QTL, but there was some genetic evidence of a QTL for response to Z11-16:OH associated with a sensory neuron membrane protein gene.

Glomerular targets of Heliothis subflexa male olfactory receptor neurons housed within long trichoid sensilla.

We used single-sensillum recordings to characterize male Heliothis subflexa antennal olfactory receptor neuron physiology in response to compounds related to their sex pheromone. The recordings were then followed by cobalt staining in order to trace the neurons' axons to their glomerular destinations in the antennal lobe. Receptor neurons responding to the major pheromone component, (Z)-11-hexadecenal, in the first type of sensillum, type-A, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of the type-A sensilla, a colocalized receptor neuron was stained that projected consistently to the posterior complex 1 (PCx1), a specific glomerulus in an 8-glomerulus complex that we call the Posterior Complex (PCx). We found that receptor neurons residing in type-B sensilla and responding to a secondary pheromone component, (Z)-9-hexadecenal, send their axons to the dorsal medial glomerulus of the MGC. As in the type-A sensilla, we found a cocompartmentalized neuron within type-B sensilla that sends its axon to a different glomerulus of the PCx4. One neuron in type-C sensilla tuned to a third pheromone component, (Z)-11-hexadecenol, and a colocalized neuron responding to (Z)-11-hexadecenyl acetate projected their axons to the anteromedial and ventromedial glomeruli of the MGC, respectively.

Inheritance of olfactory preferences I. Pheromone-mediated behavioral responses of Heliothis subflexa x Heliothis virescens hybrid male moths.

Shifts in male preference for qualitatively different pheromone blends appear to have played a fundamental role in the divergence of olfactory communication and evolution of moth species. As an initial step in documenting the genetic complexity underlying such shifts, we characterized the behavioral responses of hybrid male moths created by mating two heliothine moth species, Heliothis subflexa and Heliothis virescens. Between 67 and 96% of hybrid males flew upwind and contacted the pheromone source when presented with a blend consisting of (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-hexadecenal (Z9-16:Ald), and (Z)-11-hexadecenol (Z11-16:OH) in a 1:0.5:0.1 ratio that has previously been shown to be attractive to H. subflexa males. In addition, an H. virescens blend of Z11-16:Ald and (Z)-9-tetradecenal (Z9-14:Ald) enhanced by the addition of Z11-16:OH (in a 1:0.05:0.1 mixture) was attractive to hybrid males (26-64% source contact), but significantly fewer males reached the odor source compared to the blend containing Z9-16:Ald. A blend in which the dosage of Z9-14:Ald was doubled, however, was equally attractive (75-77% source contact) as the Z9-16:Ald-containing blend. Consecutive presentation of two blends revealed that individual hybrid males responded equally well to blends containing either Z9-14:Ald or Z9-16:Ald. Together these results suggest that in addition to Z11-16:Ald, hybrid males: (1) required either Z9-16:Ald (likeH. subflexamales) or Z9-14:Ald (like H. virescens males); (2) required the presence of Z11-16:OH (H. subflexa dominant); (3) were not adversely affected by the presence of Z11-16:Ac (H. subflexa dominant). The behavioral response phenotype of hybrid males was therefore influenced by genetic factors inherited from both parental species.

Inheritance of olfactory preferences III. Processing of pheromonal signals in the antennal lobe of Heliothis subflexa x Heliothis virescens hybrid male moths.

Pheromone-responsive olfactory interneurons were studied to determine the extent to which their physiological and morphological properties complemented the behavior and peripheral olfactory neurobiology observed in hybrid male moths created by interbreeding two species of heliothine moth, Heliothis virescens and Heliothis subflexa. Complete recordings were made from a total of 33 neurons, and 16 projection neurons (PNs) were subsequently stained with a fluorescent dye. Stained PNs tuned to pheromonal odorants had dendritic arborizations restricted to one of four olfactory glomeruli that together constituted the macroglomerular complex (MGC). As in parental males, PNs tuned to (Z)-11-hexadecenal always had an arbor in the cumulus, the largest of the MGC glomeruli. Previous neurophysiological investigations revealed that PNs with dendritic arbors restricted to the dorso-medial glomerulus (DM) of the MGC responded specifically to either (Z)-9-tetradecenal (Z9-14:Ald; H. virescens males) or (Z)-9-hexadecenal (Z9-16:Ald; H. subflexa males). Hybrid males, which responded equally well in wind tunnel tests to blends containing either Z9-14:Ald or Z9-16:Ald, had DM PNs that responded to both odorants. PNs specific for a third compound, (Z)-11-hexadecenol, required by hybrid males for behavioral activity were localized to the antero-medial MGC glomerulus (AM). Thus, neuronal activity across the cumulus, DM and AM glomeruli represented an attractive blend in hybrid males. Neurons tuned to (Z)-11-hexadecenyl acetate and Z9-14:Ald were restricted to a fourth, ventro-medial glomerulus. The across-glomerular pattern of activity associated with attractive pheromone blends was most similar to that of H. subflexa males, signifying a dominant effect of H. subflexa genes. These results indicate that the behavioral phenotype of hybrid males can be linked to underlying central olfactory characteristics.

Winging it: moth flight behavior and responses of olfactory neurons are shaped by pheromone plume dynamics.

Terrestrial odor plumes have a physical structure that results from turbulence in the fluid environment. The rapidity of insect flight maneuvers within a plume indicates that their responses are dictated by fleeting (<1 s) rather than longer (>1 s) exposures to odor imposed by physical variables that distribute odor molecules in time and space. Even though encounters with pheromone filaments are brief, male moths responding to female-produced pheromones are remarkably able to extract information relating to the biological properties of these olfactory signals. These properties include the types of molecule present and their relative abundances. Thus, peripheral and central olfactory neurons are capable of representing these biological properties of a pheromone plume within the context of a temporally irregular and unpredictable signal. The mechanisms underlying olfactory processing of these signals with respect to their biological and physical properties are discussed in the context of a behavioral framework.

Trapping Phyllophaga spp. (Coleoptera: Scarabaeidae: Melolonthinae) in the United States and Canada using sex attractants.

The sex pheromone of the scarab beetle, Phyllophaga anxia, is a blend of the methyl esters of two amino acids, L-valine and L-isoleucine. A field trapping study was conducted, deploying different blends of the two compounds at 59 locations in the United States and Canada. More than 57,000 males of 61 Phyllophaga species (Coleoptera: Scarabaeidae: Melolonthinae) were captured and identified. Three major findings included: (1) widespread use of the two compounds [of the 147 Phyllophaga (sensu stricto) species found in the United States and Canada, males of nearly 40% were captured]; (2) in most species intraspecific male response to the pheromone blends was stable between years and over geography; and (3) an unusual pheromone polymorphism was described from P. anxia. Populations at some locations were captured with L-valine methyl ester alone, whereas populations at other locations were captured with L-isoleucine methyl ester alone. At additional locations, the L-valine methyl ester-responding populations and the L-isoleucine methyl ester-responding populations were both present, producing a bimodal capture curve. In southeastern Massachusetts and in Rhode Island, in the United States, P. anxia males were captured with blends of L-valine methyl ester and L-isoleucine methyl ester.

Plasticity in central olfactory processing and pheromone blend discrimination following interspecies antennal imaginal disc transplantation.

The antennal imaginal disc was transplanted between premetamorphic male larvae of two different Lepidopteran moth species. Following adult eclosion, electrophysiological recordings were made from 33 central olfactory neurons in the antennal lobes of both Helicoverpa zea donor to Heliothis virescens recipient (Z-V) and reciprocal (V-Z) transplants. Under the influence of sensory neuron input derived from the transplanted antennal imaginal disc, most antennal lobe projection neurons (29/33) were classified as belonging to physiological categories encountered previously in donor species males. Furthermore, when stained many of these neurons had dendritic arbors restricted to donor-induced glomerular locations predicted by their physiology. However, some neurons with unexpected physiological profiles were also identified (4/33), but only in V-Z transplants. These profiles help to explain why some V-Z bilateral transplants were able to respond to both pheromone blends in flight tunnel bioassays, an unforeseen result counter to the assumption that a donor antenna develops a normal donor antennal olfactory receptor neuron complement. Stainings of several neurons in V-Z transplant males also revealed unusual morphological features including multiglomerular dendritic arbors and "incorrect" glomerular locations. These results indicate a developmental plasticity in the final dendritic arborization pattern of central olfactory neurons, including an ability to colonize and integrate inputs across topographically novel donor glomeruli, different from those found in the normal recipient antennal lobe.

Consequences of interspecies antennal imaginal disc transplantation on organization of olfactory glomeruli and pheromone blend discrimination.

The antennal imaginal disc was transplanted between male larvae of two different heliothine moth species, Heliothis virescens (HV) and Helicoverpa zea (HZ). Males of these species respond to distinct pheromone blends, have different peripheral and central olfactory neuron specificities, as well as distinct arrangements of antennal lobe olfactory glomeruli, in the specialized male macroglomerular complex (MGC). After pupal development and adult eclosion, unilateral (with one antennal disc left intact) and bilateral antennal transplant males were assayed in a wind tunnel to both species' pheromone blends to determine their ability to discriminate between the two signals. The postmetamorphic developmental effects of interspecific transplantation upon the primary olfactory centers in the moth brain were then examined in these same individuals. Behavioral tests showed that both types of unilateral transplant continued to exhibit upwind anemotactic flight to the normal recipient blend with occasional flights to the donor blend. In contrast, bilateral transplants preferred the HV pheromone blend regardless of the direction of transplant, with some males of each type also responding to the HZ blend. Neuroanatomic evaluation of the MGC revealed that the donor arrangement of MGC glomeruli was induced in 73% HZ donor to HV recipient transplants and 56% of the reciprocal transplant. Surprisingly, several V-Z bilateral transplant males responded to both HV and HZ pheromone blends and had two HV MGC structures. This behavioral outcome was unexpected, because responses to the HV blend are mediated by inputs that are normally antagonistic to HZ males and the normal HV antenna lacks olfactory receptor neurons capable of responding to the essential minor pheromone component of the HZ blend. These data indicate a plasticity in developmental pathways regulating the expression of peripheral olfactory receptor neurons and in the glomerular processing of species-specific olfactory information.

Functional divergence of spatially conserved olfactory glomeruli in two related moth species.

In different moth species, the number and spatial arrangement of olfactory glomeruli in the antennal lobe (AL) vary widely, but the spatial map within a species is thought to be invariant, making it possible to identify single glomeruli across individuals. We investigated the relationship between the physiological tuning of pheromone-selective interneurons and their association with specific, identified glomeruli in the macroglomerular complex (MGC) of the noctuid moth, Heliothis subflexa. Three odorants that are required for pheromone-source location in this species were tested individually and in blends. Recordings from 27 pheromone-specific projection neurons (PNs) indicated that the majority (48%) were selectively activated by the major pheromone component of this species, Z-11-hexadecenal (Z11-16:Ald), with 33% primarily tuned to Z-9-hexadecenal and 19% to Z-11-hexadecenol. Intracellular staining revealed that the dendrites of PNs tuned to Z11-16:Ald always branched within the largest glomerulus of the MGC, the cumulus. Similarly, each of the other two classes of PN was associated with a different 'satellite' glomerulus in the MGC. The spatial configuration of the four-glomerulus H. subflexa MGC was indistinguishable from that previously reported in the closely related species, Heliothis virescens. Hence, as these two species diverged, changes in the association of satellite MGC glomeruli with particular odorants have occurred without a measurable change in the anatomical arrangement of the glomerular array.

Defining a synthetic pheromone blend attractive to male Heliothis subflexa under wind tunnel conditions.

Heliothis subflexa males were flown in a wind tunnel to a variety of combinations of synthetic pheromone components admixed (on a filter paper disk. Blends containing (Z)-l1-hexadecenal (Z11-16:Ald, 1000 ng), (Z)-9-hexadecenal (Z9-16:Ald, 500 ng) and (Z)-11-hexadecenol (Z11-16:OH, 10-500 ng) elicited upwind flight and source contact in 52-69% of males. All these compounds have previously been isolated and identified from female H. subflexa gland extracts and volatile pheromone emissions. Males were not attracted by blends in which Z9-16:Ald was omitted (0% source contact). Similarly, blends lacking Z11-16:OH were unattractive to male H. subflexa (39% or less source contact). Males were extremely sensitive to the presence of Z11-16:OH; ever, responding in high numbers (57-69% source contact) to blends containing a dosage of 1% (10 ng) or greater Z11-16:OH. Males were unresponsive to blends in which Z9-16:Ald was replaced with a variety of dosages of (Z)-9tetradecenal, a secondary component of a closely-related congeneric species, Heliothis virescens. Another compound present in the blend emitted by con-specific females, (Z)-11-hexadecenyl acetate (Z11-16:Ac), did not inhibit H. subflexa males (69% source contact) when added to the three-component mixture (1:0.5:0.1) at a ratio of 0.1 (100 ng) with respect to Z11-16:Ald. These results indicate that Z9-16:Ald and Z11-16:OH are required in addition to Z11-16:Ald to elicit significant levels of upwind flight in H. subflexa males. The effects of Z11-16:Ac are more subtle, but at the dosage tested in these experiments, this compound does not have an antagonistic effect on upwind flight and source location by H. subflexa males.