Unraveling the Olfactory Biases of Male Euglossine Bees: Species-Specific Antennal Responses and Their Evolutionary Significance for Perfume Flowers

Male euglossine bees exhibit unique adaptations for the acquisition and accumulation of chemical compounds from “perfume flowers” and other sources. During courtship display, male bees expose perfume mixtures, presumably to convey species-specific recognition and/or mate choice signals to females. Because olfaction regulates both signal production (in males) and signal detection (in females) in this communication system, strong selective pressures are expected to act on the olfactory system, which could lead to sensory specialization in favor of an increased sensitivity to specific chemical compounds. The floral scents of euglossine-pollinated plants are hypothesized to have evolved in response to the preexisting sensory biases of their male euglossine bee pollinators. However, this has never been investigated at the peripheral olfactory circuitry of distinct pollinating genera. Here, we present a comparative analysis using electroantennography (EAG) of males across the phylogeny of 29 euglossine bee species, among them Euglossa and Eulaema species. First, we tested whether antennal responses differ among different euglossine genera, subgenera and species. Secondly, we conducted a comparative phylogenetic analysis to investigate the macroevolutionary patterns of antennal responses across the euglossine bee phylogeny. We found that antennal response profiles are very unique on the species level and differ on the subgenus and the genus level. The differences can be explained by chemical compounds typically found in the floral scent bouquets of perfume flowers and specific compounds of species either pollinated by Euglossa (e.g., ipsdienol) or Eulaema bees (e.g., (−)-(E)-carvone epoxide). Also, we detected a phylogenetic signal in mean antennal responses and found that especially at the species level of our simulation the overall antennal responses exhibit greater disparity relative to a null model of pure Brownian-motion across the phylogeny. Altogether, our results suggest that (1) euglossine bee species exhibit species-specific antennal responses that differ among euglossine genera and subgenera, (2) antennal responses diverge early after speciation events, and (3) scent composition of perfume flowers evolved in response to pollinator-mediated selection imposed by preexisting sensory biases in euglossine bees.

Identification and characterisation of female released sex pheromone components of jute semilooper, Anomis sabulifera Guenee (Lepidoptera: Noctuidae)

Aim: Identification and characterization of female released sex pheromone components of jute semilooper, Anomis sabulifera Guenee (Lepidoptera: Noctuidae) from female pheromone gland extracts. Methodology: Electroantennogram (EAG) was carried for studying the antennal response; Gas Chromatography coupled with Electro antenna Detector (GC-EAD) was conducted for studying the antennal response of eluted compounds from female pheromone gland extract; Gas Chromatography and Mas Spectrophotometry (GC-MS) was conducted for characterization or getting complete profile of compounds present in the female pheromone gland extract based on retention times. Wind tunnel assay was conducted for studying the behavioural responses of eluted compounds from the female pheromone gland extract. Results: GC-MS profile of female pheromone gland extract revealed that the GC-EAD active region constituted (6Z,9Z)-heneicosadiene, (3Z,6Z,9Z)-heneicosatriene as active compounds. Preliminary wind tunnel studies for olfactory and behavioural responses showed blend of (6Z,9Z)-heneicosadiene (3 parts) + (3Z,6Z,9Z)-heneicosatriene (1 part) enticed 60% male adults. Interpretation: (6Z,9Z)-heneicosadiene and (3Z,6Z,9Z)-heneicosatriene are likely to be active pheromone components present in female sex pheromone glands. Blending of these two compounds in precise ratio can enhance the effectiveness of pheromone and can be used as effective strategy in jute IPM. Key words: Anomis sabulifera, Jute semilooper, Noctuidae, Sex pheromone, (6Z,9Z)-heneicosadiene, (3Z,6Z,9Z)-heneicosatriene

Behavioural and antennal responses of Aedes aegypti (l.) (Diptera: Culicidae) gravid females to chemical cues from conspecific larvae

Mass trapping of gravid females represents one promising strategy for the development of sustainable tools against Aedes aegypti. However, this technique requires the development of effective odorant lures that can compete with natural breeding sites. The presence of conspecific larvae has been shown to stimulate oviposition. Hence, we evaluated the role of four major molecules previously identified from Ae. aegypti larvae (isovaleric, myristoleic, myristic [i.e. tetradecanoic], and pentadecanoic acids) on the oviposition of conspecific females, as well as their olfactory perception to evaluate their range of detection. Using flight cage assays, the preference of gravid females to oviposit in water that previously contained larvae (LHW) or containing the four larval compounds was evaluated. Then, compounds and doses inducing the highest stimulation were challenged for their efficacy against LHW. Only isovaleric acid elicited antennal response, suggesting that the other compounds may act as taste cues. Pentadecanoic acid induced significant oviposition stimulation, especially when dosed at 10 ppm. Myristoleic acid and isovaleric acid deterred oviposition at 10 and 100 ppm, while no effect on oviposition was observed with myristic acid irrespectively of the dose tested. When the four compounds were pooled to mimic larvae’s chemical signature, they favored oviposition at 1 ppm but negatively affected egg-laying at higher concentrations. When properly dosed, pentadecanoic acid and the blend of compounds may be promising lures for ovitraps as they could compete with LHW. Due to their low volatility, their effect should be further evaluated under field conditions, in addition with long-range attractants for developing effective tools against gravid females.

Heterocyclic Amine-Induced Feeding Deterrence and Antennal Response of Honey Bees

The productivity and survival of managed honey bee colonies is negatively impacted by a diverse array of interacting factors, including exposure to agrochemicals, such as pesticides. This study investigated the use of volatile heterocyclic amine (HCA) compounds as potential short-term repellents that could be employed as feeding deterrents to reduce the exposure of bees to pesticide-treated plants. Parent and substituted HCAs were screened for efficacy relative to the repellent N,N-diethyl-meta-toluamide (DEET) in laboratory and field experiments. Additionally, electroantennogram (EAG) recordings were conducted to determine the level of antennal response in bees. In video-tracking recordings, bees were observed to spend significantly less time with an HCA-treated food source than an untreated source. In a high-tunnel experiment, the HCA piperidine was incorporated in a feeding station and found to significantly reduce bee visitations relative to an untreated feeder. In field experiments, bee visitations were significantly reduced on melon flowers (Cucumis melo L.) and flowering knapweed (Centaurea stoebe L.) that were sprayed with a piperidine solution, relative to untreated plants. In EAG recordings, the HCAs elicited antennal responses that were significantly different from control or vehicle responses. Overall, this study provides evidence that HCAs can deter individual bees from food sources and suggests that this deterrence is the result of antennal olfactory detection. These findings warrant further study into structure–activity relationships that could lead to the development of short-term repellent compounds that are effective deterrents to reduce the contact of bees to pesticide-treated plants.

Inhibitory effect of thymol on pheromone-mediated attraction in two pest moth species

Plant essential oils are considered as important bio-sources for the development of natural and environmentally safe pest control tools due to their multiple modes of action on insects. In this paper we have evaluated the activity of commercially available thyme oil and its constituents thymol, carvacrol, and p-cymene, as potential disruptants of the pheromone-mediated communication in the major pest moths Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), and Grapholita molesta (Busck) (Lepidoptera: Tortricidae). In electroantennographic assays, the antennal response of males to thyme oil, thymol, and carvacrol was altered at high doses (103–104 µg), shifting the signal waveform into a biphasic negative–positive potential that caused a decay in the response. In wind tunnel assays, pheromone-mediated attraction of males of both species was interrupted in presence of thyme oil. Further trials demonstrated that thymol alone reduced the number of G. molesta and S. littoralis males landing on the pheromone source. This effect did not differ from that of thyme oil, although the latter provoked a significant reduction on downwind behavior steps in S. littoralis. Overall, our findings provide a preliminary basis for delving into the effect of thyme oil, and especially of its major constituent thymol, as potential mating disruptants of both species.

Pheromone binding protein shapes olfactory temporal resolution

Male moths are capable of orienting towards conspecific females using sex pheromones. Since pheromones are distributed as discontinuous plumes owing to air turbulence, tracking intermittent stimuli with high temporal resolution is suggested to be important for efficient localisation. Here, using a pheromone binding protein (BmPBP1) knockout silkmoth, we revealed that the loss of functional pheromone binding protein altered antennal response kinetics resulting in reduced temporal resolution to intermittent pheromone stimuli on the antennae. Behavioural analysis revealed that BmPBP1-knockout males exhibited significantly less straight walking, which occurs when detecting pheromone stimuli, especially to high frequency stimuli. Accordingly, BmPBP1-knockout males took a significantly longer time to locate pheromone sources and females than did wild-type males. Together, BmPBP1 plays a critical role in determining temporal antennal response kinetics and that an appropriate range of temporal sensory and behavioural resolutions is essential for tracking pheromone plumes for efficient pheromone source localisation in the silkmoth.

Terpenoid-Induced Feeding Deterrence and Antennal Response of Honey Bees

Multiple interacting stressors negatively affect the survival and productivity of managed honey bee colonies. Pesticides remain a primary concern for beekeepers, as even sublethal exposures can reduce bee immunocompetence, impair navigation, and reduce social communication. Pollinator protection focuses on pesticide application guidelines; however, a more active protection strategy is needed. One possible approach is the use of feeding deterrents that can be delivered as an additive during pesticide application. The goal of this study was to validate a laboratory assay designed to rapidly screen compounds for behavioral changes related to feeding or feeding deterrence. The results of this investigation demonstrated that the synthetic Nasonov pheromone and its terpenoid constituents citral, nerol, and geraniol could alter feeding behavior in a laboratory assay. Additionally, electroantennogram assays revealed that these terpenoids elicited some response in the antennae; however, only a synthetic Nasonov pheromone, citral, and geraniol elicited responses that differed significantly from control and vehicle detections.

Identification of Volatiles From Plants Infested With Honeydew-Producing Insects, and Attraction of House Flies (Diptera: Muscidae) to These Volatiles

House flies (Musca domestica L.) are mechanical vectors of food-borne pathogens including Salmonella spp., Escherichia coli O157:H7, and Shigella spp., resulting in increased risk of diarrheal disease in areas where flies are abundant. Movement of house flies into food crops may be increased by the presence of honeydew-producing insects feeding on these crops. Using gas chromatography-electroantennogram detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS), volatile odors that elicited house fly antennal response were identified from naval orange (Osbeck) (Sapindales: Rutaceae) and Marsh grapefruit (Macfad.) (Sapindales: Rutaceae) leaves infested with whitefly (Hemiptera: Aleyrodidae) and from whole faba (L.) (Fabales: Fabaceae) bean plants infested with aphids (Hemiptera: Aphididae). Volatiles identified included benzaldehyde, butyl hexanoate, β-caryophyllene, Δ3-carene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenyl acetate, myrcene, limonene, linalool, and naphthalene. This was followed by semifield bioassays of volatile blends and individual volatiles to determine house fly attraction to these volatiles. Although fly capture rates in the semifield setting were low, benzaldehyde and (Z)-3-hexenyl acetate were consistently attractive to house flies as individual compounds and as components of volatile blends.

(3R,6E)-nerolidol, a fertility-related volatile secreted by the queens of higher termites (Termitidae: Syntermitinae)

The queens of advanced social insects maintain their reproductive monopoly by using exocrine chemicals. The chemistry of these “queen pheromones” in termites is poorly known. We show that primary queens of four higher termites from the subfamily Syntermitinae (Embiratermes neotenicus, Silvestritermes heyeri, Labiotermes labralis, and Cyrilliotermes angulariceps) emit significant amounts of the sesquiterpene alcohol (E)-nerolidol. It is the dominant analyte in queen body washes; it is present on the surface of eggs, but absent in kings, workers, and soldiers. In E. neotenicus, it is also produced by replacement neotenic queens, in quantities correlated with their fertility. Using newly synthesised (3R,6E)-nerolidol, we demonstrate that the queens of this species produce only the (R) enantiomer. It is distributed over the surface of their abdomen, in internal tissues, and in the haemolymph, as well as in the headspace of the queens. Both (R) and (S) enantiomers are perceived by the antennae of E. neotenicus workers. The naturally occurring (R) enantiomer elicited a significantly larger antennal response, but it did not show any behavioural effect. In spite of technical difficulties encountered in long-term experiments with the studied species, (3R,6E)-nerolidol remains among eventual candidates for the role in queen fertility signalling.

Behavioral response of the small hive beetle, Aethina tumida (Coleoptera: Nitidulidae) to volatiles of Apicure®, a plant-based extract

Background: The small hive beetle (SHB), Aethina tumida is an invasive pest of the honey bee. Although no previous methods have led to its successful management, yeast inoculated pollen baited-traps have showed promise as quick monitoring tools. In this study, we evaluated the role of olfaction in SHB response to Apicure®, an essential oil-based biopesticide that has shown potential for the management of honey bee pests and diseases. Methods: Volatiles from Apicure® were collected using super Q adsorbent traps. Subsequent analysis was done using Gas chromatography- mass spectrophotometry (GC-MS) to ascertain the components of Apicure®. The selectivity and sensitivity of antennal receptors of A. tumida adults to the volatile compounds were determined using behavioral assays and Gas Chromatography-Electroantennodetection (GC-EAD). Results: GC-MS analysis showed that Apicure® consists of 40 compounds. GC-EAD analysis isolated 11 compounds that elicited antennal response with the SHB. Of these, linalool, camphor, geraniol and α-terpineol were confirmed to be strongly repellant, while limonene was attractive to SHB in dual-choice olfactometer assays. Conclusion: Our results demonstrate that the major components in Apicure® are mainly repellants thus prospective in disrupting the host recognition by the SHB. The product therefore can be up-scaled for the management of SHB.