Male Moth Songs Tempt Females to Accept Mating: The Role of Acoustic and Pheromonal Communication in the Reproductive Behaviour of Aphomia sociella

Valterova I (2011) Male Moth Songs Tempt Females to Accept Mating: The Role of Acoustic and Pheromonal Communication in the Reproductive Behaviour of Aphomia sociella. PLoS ONE 6(10): e26476. doi:10.1371/journal.pone.0026476 Male Moth Songs Tempt Females to Accept Mating: The Role of Acoustic and Pheromonal Communication in the Reproductive Behaviour of Aphomia sociella Jir Kindl 0 Blanka Kalinova 0 Milan C ervenka 0 Milan Jlek 0 Irena Valterova 0 Frederic Marion-Poll, AgroParisTech, France 0 1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic , Prague , Czech Republic , 2 Faculty of Electrical Engineering, Czech Technical University in Prague , Prague , Czech Republic , 3 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic , Prague , Czech Republic Background: Members of the subfamily Galleriinae have adapted to different selective environmental pressures by devising a unique mating process. Galleriinae males initiate mating by attracting females with either chemical or acoustic signals (or a combination of both modalities). Six compounds considered candidates for the sex pheromone have recently been identified in the wing gland extracts of Aphomia sociella males. Prior to the present study, acoustic communication had not been investigated. Signals mediating female attraction were likewise unknown. Methodology/Principal Findings: Observations of A. sociella mating behaviour and recordings of male acoustic signals confirmed that males initiate the mating process. During calling behaviour (stationary wing fanning and pheromone release), males disperse pheromone from their wing glands. When a female approaches, males cease calling and begin to produce ultrasonic songs as part of the courtship behaviour. Replaying of recorded courting songs to virgin females and a comparison of the mating efficiency of intact males with males lacking tegullae proved that male ultrasonic signals stimulate females to accept mating. Greenhouse experiments with isolated pheromone glands confirmed that the male sex pheromone mediates long-range female attraction. Conclusion/Significance: Female attraction in A. sociella is chemically mediated, but ultrasonic communication is also employed during courtship. Male ultrasonic songs stimulate female sexual display and significantly affect mating efficiency. Considerable inter-individual differences in song structure exist. These could play a role in female mate selection provided that the female's ear is able to discern them. The A. sociella mating strategy described above is unique within the subfamily Galleriinae. - Funding: This research was supported financially by Grant #2B06007 (Ministry of Education, Youth and Sports of the Czech Republic) and by Research Project #Z4055905 (Academy of Sciences of the Czech Republic). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Moths of three main superfamilies (Pyralidoidea, Geometroidea, and Noctuidea) have tympanal ears sensitive to ultrasound [1,2]. The ears probably developed in response to pressure from predators (bats) [3,4]. Following ear development, many moth species developed sound producing organs and ultrasound emission became a part of sexual communication in some species [1,57]. Members of the subfamily Galleriinae have adapted to different selective environmental pressures by devising an unusual mating system, where males initiate mating and attract females using either chemical or acoustic signals (Table 1). Three types of ultrasonic communication signals have been reported in Galleriinae to date: i) songs to attract mates (the lesser wax moth, Achroia grisella) [814], ii) courtship songs for mate acceptance/recognition (the greater wax moth, Galleria mellonella and the African sugarcane borer Eldana saccharina [1520]), and iii) rival songs employed during male signalling (A. grisella, G. mellonella, E. saccharina) [9,18,2123]. Ultrasonic emission in Galleriinae is associated with wing movements that induce high-frequency oscillations of tegular tymbals [1,23]. The composition of male sex pheromones, tymbal morphology, and the sonic patterns, frequencies, and intensities of ultrasonic communication are all species-specific (Table 1). Aphomia sociella L. (Pyralidae, Galleriinae) is becoming an economically important pest in bumblebee mass-production facilities. Recently, six candidate compounds for the sex pheromone have been identified in the wing gland extracts and emanations of A. sociella males [24]. However, nothing is yet known about the signals that mediate female attraction in this species. The present study addresses the following questions: i) How do A. sociella males attract females? Do they use sex pheromones, acoustic signals, or a combination of the two? ii) What is the morphology of the sound-producing tymbals? iii) What are the physical parameters of acoustic signals (sonic patterns, frequencies, and intensities)? iv) How sensitive is the female ear? And, consequently, what is the communication range of ultrasonic 1 Z :O signalling in A. sociella? v) Does ultrasonic communication affect mating efficiency? vi) How do our data fit into the evolution pattern of chemical and acoustic communication in Galleriinae? Materials and Methods 1. Insects Bombus terrestris nests were colonised with A. sociella and left to overwinter. In the spring, larvae were allowed to pupate. After eclosion, the moths were segregated in individual plastic containers stoppered by moistened cotton plugs. Pilot experiments designed to determine the peak of mating activity were performed during the entire circadian period. Semi-field experiments were conducted after dusk. In the laboratory, the moths were kept in continuous light and used in experiments after 1 hour of acclimation in the dark. Virgin moths (24 days old) were utilised. 2. SEM of Tegular Morphology Male and female tegulae were dissected and sonicated for 3 minutes to remove scales. The tegulae were then dehydrated in a series of 30100% aqueous alcohol and 100% acetone. Dehydrated tegulae were mounted on aluminium stubs, coated with 300 nm of gold:palladium (60:40) alloy, and visualised using a JEOL 6380 LV scanning electron microscope. 3. A. sociella Mating Behaviour Experiments designed to understand individual phases of the A. sociella mating behaviour were performed under red light and at ambient temperature/humidity. During photophase, pairs of insects were placed in individual round dishes (25 cm in diameter) made of wire mesh (one pair per dish). The same insects were then observed during scotophase. Ultrasonic signalling was detected using a bat detector (heterodyne Mini 3 Bat Detector, Ultra Sound Advice, UK) set at 80 kHz (the dominant frequency of Aphomia sociella ultrasonic songs see bel (...truncated)