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.

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.

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.

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.

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.

Attraction Versus Capture II: Efficiency of the BG-Sentinel Trap Under Semifield Conditions and Characterizing Response Behaviors of Male Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) is an important vector of viruses causing dengue, Zika, chikungunya, and yellow fever and as such presents a serious threat to public health in tropical regions. Control programs involving 'rear and release' of modified male Ae. aegypti are underway and require effective trapping methods for surveillance of both the released insects and the impacted wild mosquito population. The BG-Sentinel trap (BGS) is widely used in Ae. aegypti surveillance but its level of efficiency, that is, what proportion of the mosquitoes encountering the trap are captured, is unknown. This is especially true for male mosquitoes, the behavior of which is incompletely understood. We tested the efficiency of two versions of the BGS for capturing male Ae. aegypti under semifield conditions with and without CO2 and a human skin odor mimic lure and with these baits combined. A navy-blue BGS trap emitting CO2 and a human skin odor mimic captured 18% of the released male Ae. aegypti, with a capture efficiency of 9 % (of the total encounters with the trap). Male Ae. aegypti had multiple encounters with the BGS that did not result in capture; they crossed over the trap entrance without being captured or landed on the sides of the trap. Swarming behavior around the BGS was also recorded, even when only a visual cue was present. Understanding male Ae. aegypti behaviors during an encounter with the BGS can inform improvement of trap design and therefore capture efficiency for surveillance in control programs.

Attraction Versus Capture: Efficiency of BG-Sentinel Trap Under Semi-Field Conditions and Characterizing Response Behaviors for Female Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) is an important vector of viruses causing dengue, Zika, chikungunya, and yellow fever and as such is a threat to public health worldwide. Effective trapping methods are essential for surveillance of both the mosquito species and disease presence. The BG-Sentinel (BGS) is a widely used to trap Ae. aegypti but little is known of its efficiency, i.e., what proportion of the mosquitoes encountering the trap are captured. The first version of the BGS trap was predominantly white, and the current version is mostly navy blue. While this trap is often deployed without any olfactory lure, it can also be deployed with CO2 and/or a human skin odor mimic lure to increase capture rates. We tested the efficiency of capturing Ae. aegypti under semi-field conditions for the original white version without lures as well the blue version with and without various lure combinations. None of the configurations tested here captured 100% of the mosquitoes that encountered the trap. A navy-blue trap emitting CO2 and a skin odor mimic produced the highest capture (14% of the total insects in the semi-field cage), but its capture efficiency was just 5% (of mosquitoes encountering the trap). Mosquitoes often had multiple encounters with a trap that did not result in capture; they crossed over the trap entrance without being captured or landed on the sides of the trap. Understanding these behaviors and the factors that induce them has the potential to suggest improvement in trap design and therefore capture efficiency.

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.

Comparison of Attraction and Trapping Capabilities of Bucket- and Delta-Style Traps With Different Pheromone Emission Rates for Gypsy Moths (Lepidoptera: Erebidae): Implications for Understanding Range of Attraction and Utility in Surveillance.

Delta- and bucket-style (Universal or Unitrap) traps baited with 1 standard survey lure and 1/3 and 3 lures were compared for their attractiveness and trapping efficiencies for gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae), males. With bucket traps, the numbers of males attracted to within 2 m of traps and the proportion of these actually captured were identical among the three doses although the percentage of attracted males actually captured in bucket traps was low, less than 15%. A three-lure delta trap attracted about 70% more males than traps with the two lower doses. Capture efficiencies were above 80% for 1/3- and one-lure traps and about 60% for traps baited with three lures. The number of males captured in delta traps was equivalent for the three doses although our observations also suggest that a delta trap baited with three lures drew males from a wider range than lower dose lures and therefore would be a more sensitive trap for detecting incipient populations. We also noted that males tended to arrive in clusters, suggesting that attraction over moderate distances requires periods when the wind direction is fairly constant. This observation coupled with the great variability in the direction of male arrival to the traps also suggests that important changes in the area of influence of the plume are driven in such forested areas by slower but greater changes in wind direction compared with open habitats.

Mating Disruption of the Navel Orangeworm (Lepidoptera: Pyralidae) Using Widely Spaced, Aerosol Dispensers: Is the Pheromone Blend the Most Efficacious Disruptant?

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is a key pest of almonds and pistachios and is sometimes controlled using mating disruption as part of a program of integrated management. The formulation used has a single, nonattractive compound [(11Z,13Z)-hexadecadienal] as the active ingredient that is emitted from timed aerosol dispensers. This study compared this nonattractive, single-compound formulation with two aerosol formulations also containing two additional compounds [(11Z,13Z)-hexadecadien-1-ol and (3Z,6Z,9Z,12Z,15Z)-tricosapentaene] that are found in the pheromone glands, and that in combination with the aldehyde are attractive in wind-tunnel and field-attraction trials. An experiment in pistachios found 97% to 99% suppression of males captured in female-baited traps and 82-93% suppression of mating in sentinel females. Both assays revealed a trend to greater suppression by the more complete pheromone formulations. In almonds, where the abundance of navel orangeworm was lower, all three formulations suppressed males captured in traps and mating in sentinel females by >99%. Each of the formulations significantly reduced damage to Nonpareil almonds. In almonds, there were no significant differences among the formulations in disruption of sexual communication or in damage. These findings suggest that it may be possible to make mating disruption more cost-effective and to achieve higher levels of mating disruption by using attractive aerosol formulations to reduce the number of dispenser per ha. Such a formulation, however, would be more expensive to register in the United States than pheromones meeting the definition of straight-chain lepidopteran pheromone, including the currently used aldehyde-only formulation.

Use of habitat odour by host-seeking insects.

Locating suitable feeding or oviposition sites is essential for insect survival. Understanding how insects achieve this is crucial, not only for understanding the ecology and evolution of insect-host interactions, but also for the development of sustainable pest-control strategies that exploit insects' host-seeking behaviours. Volatile chemical cues are used by foraging insects to locate and recognise potential hosts but in nature these resources usually are patchily distributed, making chance encounters with host odour plumes rare over distances greater than tens of metres. The majority of studies on insect host-seeking have focussed on short-range orientation to easily detectable cues and it is only recently that we have begun to understand how insects overcome this challenge. Recent advances show that insects from a wide range of feeding guilds make use of 'habitat cues', volatile chemical cues released over a relatively large area that indicate a locale where more specific host cues are most likely to be found. Habitat cues differ from host cues in that they tend to be released in larger quantities, are more easily detectable over longer distances, and may lack specificity, yet provide an effective way for insects to maximise their chances of subsequently encountering specific host cues. This review brings together recent advances in this area, discussing key examples and similarities in strategies used by haematophagous insects, soil-dwelling insects and insects that forage around plants. We also propose and provide evidence for a new theory that general and non-host plant volatiles can be used by foraging herbivores to locate patches of vegetation at a distance in the absence of more specific host cues, explaining some of the many discrepancies between laboratory and field trials that attempt to make use of plant-derived repellents for controlling insect pests.

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.

Multi-Cue Integration: How Female Mosquitoes Locate a Human Host.

To reproduce, the female yellow fever mosquito has to find a human host. There are many potential cues available to guide such navigation: exhaled carbon dioxide, a plethora of skin odors, the host's visual and heat signatures and, close by, moisture. Recent work is shedding new light on how these are integrated by the mosquito in targeting a human host.

Modeling Optimal Strategies for Finding a Resource-Linked, Windborne Odor Plume: Theories, Robotics, and Biomimetic Lessons from Flying Insects.

Male moths locate females by navigating along her pheromone plume, often flying hundreds of meters en route. As the first male to find a calling female is most apt to be her mate, this can be termed "a race to find the female" and it is assumed to be under strong selective pressure for efficiency and rapidity. Locating a distant, odor-linked resource involves two strategies. The first is to contact the outer envelope of the odor plume. When wind direction is relatively invariant, the plume stretches and then crosswind flights may be favored, although when wind direction shifts over 60°, upwind and downwind paths may be optimal. Alternatively, the path may be random with respect to the direction of wind flow, with periodic changes in direction, as in either Lévy or Random Walks. After first detecting the pheromone, a second strategy follows: moths navigate along the plume by heading upwind when the pheromone is detected, with crosswind casting to re-establish contact if the plume is lost. This orientation path is not straightforward in nature, however, because atmospheric turbulence fragments the plume, thereby creating large odor gaps. Furthermore, a shifting wind direction can lead the responder out of the plume. One way to explore which strategies are optimal for enabling initial contact with the plume and subsequent navigation is through modeling of plumes' dispersal and of insects' flight strategies. Our simulations using the flight characteristics of the male gypsy moth (Lymantria dispar) suggest that search strategies similar to Lévy Walks are most apt to result in a high probability of contact with plumes. Although a searching trajectory aimed predominately crosswind performed almost as well as those with a random orientation when wind direction was relatively stable, downwind biased trajectories were least successful. A random orientation with respect to immediate wind flow, as used in our simulations of Lévy and Random Walks, seems optimal both for initial discovery of the plume and likelihood of locating an odor source. In the two available direct field observations, moths adopted a random orientation with respect to concurrent wind direction.

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.

Navigation: many senses make efficient foraging paths.

Desert ants have a sequence of optimized behaviours that allow them to forage efficiently. Recent work shows that after using navigational memories to reach previously rewarding areas, ants follow long crosswind sweeps that appear adapted for encountering odour plumes.