Navigation Along Windborne Plumes of Pheromone and Resource-Linked Odors.

Many insects locate resources such as a mate, a host, or food by flying upwind along the odor plumes that these resources emit to their source. A windborne plume has a turbulent structure comprised of odor filaments interspersed with clean air. As it propagates downwind, the plume becomes more dispersed and dilute, but filaments with concentrations above the threshold required to elicit a behavioral response from receiving organisms can persist for long distances. Flying insects orient along plumes by steering upwind, triggered by the optomotor reaction. Sequential measurements of differences in odor concentration are unreliable indicators of distance to or direction of the odor source. Plume intermittency and the plume's fine-scale structure can play a role in setting an insect's upwind course. The prowess of insects in navigating to odor sources has spawned bioinspired virtual models and even odor-seeking robots, although some of these approaches use mechanisms that are unnecessarily complex and probably exceed an insect's processing capabilities.

Efficiency of the CO2 -baited omni-directional Fay-Prince trap under semi-field conditions and characterizing response behaviours for the yellow fever mosquito, Aedes aegypti.

Aedes aegypti (L.) (Diptera: Culicidae) is a vector of several serious disease-causing viruses including Dengue, Zika, chikungunya and yellow fever. Effective and efficient trapping methods are essential for meaningful mosquito population and disease-presence surveillance and ultimately, vector control. The Fay-Prince trap (FPT) was developed in the late 1960s as a daytime visual trap for male Ae. aegypti. Since then, its use has been expanded into the trapping of female Ae. aegypti, Aedes albopictus Skuse, other Ae. spp., and Culex spp. The efficiency of the FPT alone and with CO2 was tested under semi-field conditions and the behaviour of responding female Ae. aegypti was characterized. The mean capture efficiency of the FPT with CO2 per 30 min in the greenhouse was 3.07% and the capture rate from the total number of mosquitoes in our semi-field setup was slightly higher at 4.45%. Understanding the behaviours that mosquitoes exhibit during their encounter with particularly a visual trap may recommend trap improvements and contributes to our understanding of host-seeking behaviour and how it might be exploited.

Do Helicoverpa armigera Moths Signal Their Fecundity by Emission of an Antagonist?

Female Helicoverpa armigera emit a pheromone, comprised of a 98:2 ratio of (Z)-11-hexadecenal to (Z)-9-hexadecenal, to attract males. It has been proposed that "immature" female H. armigera modulate attraction of males by emitting an antagonist, (Z)-11-hexadecenol, along with pheromone during the first two nights of calling. However, it is unclear why females would call and simultaneously release pheromone and an antagonist. We conducted observations of female calling during the first five nights after adult emergence to determine periodicity. We also measured the relative abundance of (Z)-11-hexadecenol to the major component, (Z)-11-hexadecenal, on the surface of the gland of calling females and compared it to the ratio of these two compounds inside the gland over the first three nights after adult emergence to determine how much antagonist may be released. We found that young females (< 1-d-old) are unlikely to call and, based on the relative proportion of (Z)-11-hexadecenol on the gland surface, even if they did call would be unlikely to release sufficient (Z)-11-hexadecenol to diminish male attraction.

Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes.

Carbon dioxide (CO(2)) present in exhaled air is the most important sensory cue for female blood-feeding mosquitoes, causing activation of long-distance host-seeking flight, navigation towards the vertebrate host and, in the case of Aedes aegypti, increased sensitivity to skin odours. The CO(2) detection machinery is therefore an ideal target to disrupt host seeking. Here we use electrophysiological assays to identify a volatile odorant that causes an unusual, ultra-prolonged activation of CO(2)-detecting neurons in three major disease-transmitting mosquitoes: Anopheles gambiae, Culex quinquefasciatus and A. aegypti. Importantly, ultra-prolonged activation of these neurons severely compromises their ability subsequently to detect CO(2) for several minutes. We also identify odours that strongly inhibit CO(2)-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO(2)-like activity and thus has potential use as a lure in trapping devices. Analysis of responses to panels of structurally related odours across the three mosquitoes and Drosophila, which have related CO(2)-receptor proteins, reveals a pattern of inhibition that is often conserved. We use video tracking in wind-tunnel experiments to demonstrate that the novel ultra-prolonged activators can completely disrupt CO(2)-mediated activation as well as source-finding behaviour in Aedes mosquitoes, even after the odour is no longer present. Lastly, semi-field studies demonstrate that use of ultra-prolonged activators disrupts CO(2)-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO(2)-response-modifying odours offer powerful instruments for developing new generations of insect repellents and lures, which even in small quantities can interfere with the ability of mosquitoes to seek humans.

Simulation Modeling to Interpret the Captures of Moths in Pheromone-Baited Traps Used for Surveillance of Invasive Species: the Gypsy Moth as a Model Case.

When pheromone traps are used for detection of an invasive pest and then delimitation of its distribution, an unresolved issue is the interpretation of failure to capture any target insects. Is a population present but not detected, a so-called false negative? Using the gypsy moth (Lymantria dispar) as an exemplar, we modeled the probability of males being captured in traps deployed at densities typical for surveillance (1 per 2.6 km2 or 1 per mi2) and delimitation (up to 49 per 2.6 km2). The simulations used a dynamic wind model generating a turbulent plume structure and varying wind direction, and a behavior model based on the documented maneuvers of gypsy moths during plume acquisition and along-plume navigation. Several strategies of plume acquisition using Correlated Random Walks were compared to ensure that the generated dispersions over three days were not either overly clumped or ranged many km. Virtual moths were released into virtual space with patterns mimicking prior releases of gypsy moth males in Massachusetts at varying distance from a baited trap. In general, capture rates of virtual and real moths at varying trap densities were similar. One application of this approach was to estimate through bootstrapping the probabilities of not detecting populations having densities ranging from 1 to 100 moths per 2.6 km2 and using traps that varied from 25 to 100 % in their efficiencies of capture. Low-level populations (e.g., 20-30 per 2.6 km2) often were not detected with one trap per 2.6 km2, especially when traps had low efficiencies.

Tarsi of Male Heliothine Moths Contain Aldehydes and Butyrate Esters as Potential Pheromone Components.

The Noctuidae are one of the most speciose moth families and include the genera Helicoverpa and Heliothis. Females use (Z)-11-hexadecenal as the major component of their sex pheromones except for Helicoverpa assulta and Helicoverpa gelotopoeon, both of which utilize (Z)-9-hexadecenal. The minor compounds found in heliothine sex pheromone glands vary with species, but hexadecanal has been found in the pheromone gland of almost all heliothine females so far investigated. In this study, we found a large amount (0.5-1.5 µg) of hexadecanal and octadecanal on the legs of males of four heliothine species, Helicoverpa zea, Helicoverpa armigera, H. assulta, and Heliothis virescens. The hexadecanal was found on and released from the tarsi, and was in much lower levels or not detected on the remaining parts of the leg (tibia, femur, trochanter, and coxa). Lower amounts (0.05-0.5 µg) of hexadecanal were found on female tarsi. This is the first known sex pheromone compound to be identified from the legs of nocturnal moths. Large amounts of butyrate esters (about 16 µg) also were found on tarsi of males with lower amounts on female tarsi. Males deposited the butyrate esters while walking on a glass surface. Decapitation did not reduce the levels of hexadecanal on the tarsi of H. zea males, indicating that hexadecanal production is not under the same neuroendocrine regulation system as the production of female sex pheromone. Based on electroantennogram studies, female antennae had a relatively high response to hexadecanal compared to male antennae. We consider the possible role of aldehydes and butyrate esters as courtship signals in heliothine moths.

Early quality assessment lessens pheromone specificity in a moth.

Pheromone orientation in moths is an exemplar of olfactory acuity. To avoid heterospecific mating, males respond to female-produced blends with high specificity and temporal resolution. A finely tuned sensory to projection neuron network secures specificity, and this network is thought to assess pheromone quality continually during orientation. We tested whether male moths do indeed evaluate each pheromone encounter and surprisingly found that male European corn borer moths instead generalize across successive encounters. Although initially highly ratio specific, once "locked on" to the pheromone plume the acceptable ratio can vary widely, and even unattractive blends can become attractive. We further found that this "mental shortcut" may be a consequence of the fact that sensory neurons exposed to frequent encounters do not reliably encode blend ratios. Neurons tuned to either of the two pheromone components adapt differentially in plumes containing the preferred blend ratio (97:3) and cause the olfactory sensory signal to "evolve," even in narrowly tuned pheromonal circuits. However, apparently the brain interprets these shifting signals as invariant "gestalts." Generalization in pheromone perception may mitigate stabilizing selection and allow introgression between sympatric strains, such as in the European corn borer, that otherwise appear isolated by pheromonal differences. Generalization may also be important in responses to general odorants, as circuits underlying these display vast sensitivity differences, complex interactions, and temporal intricacies.

Waiting with bated breath: opportunistic orientation to human odor in the malaria mosquito, Anopheles gambiae, is modulated by minute changes in carbon dioxide concentration.

Females of the malaria mosquito, Anopheles gambiae, predominantly obtain blood meals within human dwellings. Being highly anthropophilic, human skin odor offers a reliable, host-specific cue, but the challenge posed by pervasive human odor found indoors from used clothing, bedding etc. remains unclear. Anopheles gambiae spends much of its adult life indoors, constantly exposed to human odor even when dwellings are unoccupied. In landing assays, we found that female mosquitoes respond very weakly to human skin odor alone, suggesting that, alone, it is an ineffective landing cue. Landing, however, was dramatically increased by addition of carbon dioxide at a range of concentrations above ambient. Indeed, this effect was seen even when carbon dioxide was just 0.015% above ambient within the assay cage. The synergistic effect of added carbon dioxide quickly waned, thereby facilitating a highly adaptive "sit-and-wait" ambush strategy, wherein females ignore persistent human odor until a living human is present. Unexpectedly, landing rates in the presence of added carbon dioxide were almost as robust during daytime, when An. gambiae has previously been assumed inactive, possibly facilitating opportunistic feeding at times of day when human dwellings are occupied intermittently. We suggest earlier studies that showed strong upwind flight behavior toward human odor alone could, in fact, have been demonstrating orientation toward a human dwelling rather than toward a living human. This new interpretation of how human odors mediate upwind orientation and landing in An. gambiae is discussed.

Following in their footprints: cuticular hydrocarbons as overwintering aggregation site markers in Hippodamia convergens.

The convergent lady beetle (Hippodamia convergens) forms large overwintering aggregations at revisited montane microsites far removed from their summer foraging grounds. Although orientation responses to visual and altitudinal features of habitat can explain the arrival of migrants at the general overwintering macrosite, the role that pheromones play in the accumulation of individuals in inconspicuous hibernacula microsites is not fully understood. Through two-choice bioassays and gas chromatography and mass spectrometry, we found that H. convergens orient towards hydrocarbons previously deposited on their walking surfaces by conspecifics. n-Tricosane (C23) is primarily responsible for this chemically-mediated orientation. Footprint extracts, as well as C23 alone, induce the eventual accumulation in the field of migrant H. convergens at artificial hibernacula, confirming their probable role as aggregation signals. Aggregations persisted over many days when footprint extracts were applied in conjunction with the previously identified 2-isobutyl-3-methoxypyrazine (IBMP) aggregation pheromone. The C23 hydrocarbon functions as a pheromone that interacts with responses to methoxypyrazines to effectively mediate formation of persistent aggregations of diapausing conspecifics at specific microsites. Also discussed is the potential effect that C23 has as a persistent scent marker in establishing the traditional use of hibernacula.

Wind Tunnels and Airflow-Driven Assays: Methods for Establishing the Cues and Orientation Mechanisms That Modulate Female Mosquito Attraction to Human Hosts.

Understanding how female mosquitoes find a prospective host is crucial to developing means that can interfere with this process. Many methods are available to researchers studying cues and orientation mechanisms that modulate female mosquito attraction to hosts. Behaviors that can be monitored with these assays include activation, taking flight, upwind flight along an odor plume (optomotor anemotaxis), close approach to the stimulus (including hovering), and landing. Video recording can three-dimensionally document flight tracks and can correlate overall distribution patterns and moment-to-moment movements with odor contact and the presence of nearby cues such as a visual target. Here, we introduce mosquito host-seeking behaviors and methods to study them: wind tunnels (which allow orientation in free-flight), airflow-driven assays (using either tethered mosquitoes or small assay chambers that permit flight but also often dictate walking orientation), and still-air assays (wherein in odor concentration and spatial distribution are the orientation cues). We also describe factors that affect the assays and provide assay design considerations.

Defensive allomones function as aggregation pheromones in diapausing Ladybird Beetles, Hippodamia convergens.

Identification of the stimuli responsible for the formation of an aggregation can be used to distinguish between social and non-social aggregations and help in the process of identifying the adaptive benefits of the gregarious behavior. The convergent ladybird beetle, Hippodamia convergens, forms dense aggregations during winter diapause. The mechanisms of conspecific attraction and hibernacula site selection of H. convergens are not well understood. In laboratory and field bioassays, we evaluated the role of three defensive compounds in the formation of H. convergens aggregations. Diapausing H. convergens aggregated within the section of an arena exposed to alkylmethoxypyrazines. 2-Isobutyl-3-methoxypyrazine (IBMP) caused the strongest aggregative effect. Beetles also aggregated to some doses of 2-sec-butyl-3-methoxypyrazine, but appeared to be repelled at higher doses. A third constituent, 2-isopropyl-3-methoxypyrazine, generally had little effect on the distribution of beetles, although the highest dose tested was repellent. Beetles also aggregated to a blend of these alkylmethoxypyrazines at their natural ratio. During fall migration to overwintering sites, more beetles aggregated in artificial hibernacula baited with IBMP, confirming its function as an aggregation pheromone. These three pyrazines also function as warning odors that, in conjunction with other aposematic displays (contrasting red and black coloration, gregarious behavior, reflex bleeding), contribute to the multi-modal, anti-predatory defense of coccinellid beetles and some other arthropods. Confirmation of the role of some alkylmethoxypyrazines in coccinellid aggregations suggests that these defensive allomones have been co-opted for intraspecific communication.

The plume also rises: trajectories of pheromone plumes issuing from point sources in an orchard canopy at night.

The trajectories of pheromone plumes in canopied habitats, such as orchards, have been little studied. We documented the capture of male navel orangeworm moths, Amyelois transitella, in female-baited traps positioned at 5 levels, from ground level to the canopy top, at approximately 6 m above ground, in almond orchards. Males were captured in similar proportions at all levels, suggesting that they do not favor a particular height during ranging flight. A 3-D sonic anemometer was used to establish patterns of wind flow and temperature at 6 heights from 2.08 to 6.65 m in an almond orchard with a 5 m high canopy, every 3 h over 72 h. The horizontal velocity of wind flow was highest above the canopy, where its directionality also was the most consistent. During the time of A. transitella mating (0300-0600), there was a net vertical displacement upward. Vertical buoyancy combined with only minor reductions in the distance that plumes will travel in the lower compared to the upper canopy suggest that the optimal height for release of pheromone from high-release rate sources, such as aerosol dispensers ("puffers"), that are deployed at low densities (e.g., 3 per ha.) would be at mid or low in the canopy, thereby facilitating dispersion of disruptant throughout the canopy. Optimal placement of aerosol dispensers will vary with the behavioral ecology of the target pest; however, our results suggest that current protocols, which generally propose dispenser placement in the upper third of the canopy, should be reevaluated.

Observations on the flight paths of the day-flying moth Virbia lamae during periods of mate location: do males have a strategy for contacting the pheromone plume?

1. To maximize the probability of rapid contact with a female's pheromone plume, the trajectories of male foraging flights might be expected to be directed with respect to wind flow and also to be energetically efficient. 2. Flights directed either upwind, downwind, or crosswind have been proposed as optimal strategies for rapid and/or energetically efficient plume contact. Other possible strategies are random and Lévy walks, which have trajectories and turn frequencies that are not dictated by the direction of wind flow. 3. The planar flight paths of males of the day-active moth Virbia lamae were recorded during the customary time of its sexual activity. 4. We found no directional preference in these foraging flights with respect to the direction of contemporaneous wind flow, but, because crosswind encompasses twice the possible orientations of either upwind or downwind, a random orientation is in effect a de facto crosswind strategy. 5. A crosswind preference should be favoured when the plume extends farther downwind than crosswind, and this strategy is realized by V. lamae males by a random orientation of their trajectories with respect to current wind direction.

Verifying the efficiency of the Biogents Sentinel trap in the field and investigating microclimatic influences on responding Aedes aegypti behavior.

Successful surveillance and control of mosquito arbovirus vectors requires effective and sensitive trapping methods for adult insects. The Biogents Sentinel (BGS) trap is widely used for mosquito trapping but has low capture efficiency for both female and male Aedes aegypti under semi-field conditions. Efficiency refers to the proportion of mosquitoes that are trapped of those encountering the trap. We verified the efficiency of the BGS under field conditions in suburban Riverside, California, U.S.A., following our previous work determining the efficiency under semi-field conditions in Cairns, Northern Australia. The efficiency of the BGS with CO2 and a human skin odor mimic (BG-Lure) for both Ae. aegypti sexes in the field was 9%. This closely aligns with the results of our previous study, the efficiency for females being 5% and males being 9%. In the present study microclimatic conditions were monitored and capture occurred during periods of significantly lower mean temperature. There were no discernible changes in wind directionality or strength in the 60 s leading up to mosquito capture by the BGS. Our results support our previous findings that capture efficiency of the BGS for Ae. aegypti is low.

Compounds from human odor induce attraction and landing in female yellow fever mosquitoes (Aedes aegypti).

The female Aedes aegypti mosquito is a vector of many human diseases such as yellow fever, dengue, and Zika. Transmission of these viruses occurs when an infected female mosquito locates a suitable human host, alights, and blood feeds. Aedes aegypti use human-emitted odors, as well as heat and visual cues, for host location. However, none of the previously identified human-produced compounds induce significant orientation and landing on a human host. Here we show that female yellow fever mosquitoes orient to and land on a mixture of compounds identified from human skin rubbings. Using odor collection, extraction, a two-choice, bioassay-guided fractionation, and chemical analysis, we identified mixtures of 2-ketoglutaric acid and L-lactic acid as landing attractants for female Ae. aegypti. The mixture of pyruvic acid and L-lactic acid were also found to be weakly attractive. Using ratio-response assays, we found that the attraction and alighting behaviors of the mosquitoes were directly related to the ratio of these compounds presented on the surface of the glass assay beads, suggesting that these compounds could mediate landing on a human host even at sub-nanogram dosages. The newly identified compounds fill a gap in our knowledge of odor-mediated attraction of Ae. aegypti and may lead to the development of new attractant-based mosquito control tactics.

Primacy of Human Odors Over Visual and Heat Cues in Inducing Landing in Female Aedes aegypti Mosquitoes.

Although human skin odor is thought to be the cue that anthropophilic mosquitoes use to discriminate us from other potential hosts, the precise details of how they use skin odor to find and land on a human is unclear. We found that Aedes aegypti land on a source of skin odor without a co-located visual cue. By collecting human odor on glass beads and using identical glass beads to visually conceal skin odor and heat cues, we were able to study mosquito landing on skin odor, heat, and visual cues separately. Landing is necessary for blood feeding which is a required behavior for the Aedes aegypti life cycle as well as the behavior responsible for the epidemiological impact of mosquitoes. Therefore, we consider it to be the diagnostic measure of the importance of a host cue. In two-choice tests, a skin odor source had the highest valence for landing, followed by a combination of heat and a visual cue, and finally heat and visual cues presented separately. We also measured the durations of the landings, though no significant differences were found. Supplementary Information: The online version contains supplementary material available at 10.1007/s10905-022-09796-2.

Animal migration: seasonal reversals of migrant moths.

A recent study has found that, as migrating silver Y moths pass high overhead above central England in the spring, their headings were generally aimed towards north---a reversal of direction relative to that of autumn migrants. The silver Y must detect its direction of movement, likely by a magnetic sense which must reverse with the season.

Insect migration: do migrant moths know where they are heading?

Moth migration has been assumed to involve hitching a ride in favorable winds. A new study has shown that silver Y moths migrate only on nights when winds would displace them southward, implying that they detect their direction of movement while airborne, likely by a magnetic sense.

Moment-to-moment flight manoeuvres of the female yellow fever mosquito (Aedes aegypti L.) in response to plumes of carbon dioxide and human skin odour.

Odours are crucial cues enabling female mosquitoes to orient to prospective hosts. However, their in-flight manoeuvres to host odours are virtually unknown. Here we analyzed in 3-D the video records of female Aedes aegypti mosquitoes flying in a wind tunnel in response to host odour plumes that differed in spatial structure and composition. Following a brief (~0.03 s) encounter with CO(2), mosquitoes surged upwind and, in the absence of further encounters, counterturned without displacing upwind. These patterns resemble moth responses to encounter and loss of a filament of pheromone. Moreover, CO(2) encounters induced a highly regular pattern of counterturning across the windline in the horizontal (crosswind) and vertical planes, causing the mosquito to transect repeatedly the area where CO(2) was previously detected. However, despite the rapid changes across all three axes following an encounter with CO(2), the angular velocities remained remarkably constant. This suggests that during these CO(2)-induced surges mosquitoes stabilize flight through sensors, such as the halteres and Johnston organs, sensitive to Coriolis forces. In contrast to the instantaneous responses of the mosquito CO(2), a brief encounter with a filament of human skin odour did not induce a consistent change in mosquito flight. These differential responses were reflected in further experiments with broad plumes. A broad homogeneous plume of skin odour induced rapid upwind flight and source finding, whereas a broad filamentous plume of skin odour lowered activation rates, kinetic responses and source finding compared with homogeneous plumes. Apparently, yellow fever mosquitoes need longer continuous exposure to complex skin-odour blends to induce activation and source finding.

In addition to cryptochrome 2, magnetic particles with olfactory co-receptor are important for magnetic orientation in termites.

The volatile trail pheromone is an ephemeral chemical cue, whereas the geomagnetic field (GMF) provides a stable positional reference. However, it is unclear whether and how the cryptic termites perceive the GMF for orientation in light or darkness until now. Here, we found that the two termite species, Reticulitermes chinensis and Odontotermes formosanus, use the GMF for orientation. Our silencing cryptochrome 2 (Cry2) impaired magnetic orientation in white light but had no significant impact in complete darkness, suggesting that Cry2 can mediate magnetic orientation in termites only under light. Coincidentally, the presence of magnetic particles enabled the magnetic orientation of termites in darkness. When knock-downing the olfactory co-receptor (Orco) to exclude the effect of trail pheromone, unexpectedly, we found that the Orco participated in termite magnetic orientation under both light and darkness. Our findings revealed a novel magnetoreception model depending on the joint action of radical pair, magnetic particle, and olfactory co-receptor.