4091 INTRODUCTION The general female preference for and orientation toward male advertisements broadcast at high signal intensity is well known in the contexts of neuroethology and sexual selection (Andersson, 1994; Bradbury and Vehrencamp, 1998). Minimum thresholds of signal intensity that are necessary for evoking a neural or behavioral response in females normally exist. Moreover, females often exhibit higher levels of response as signal intensity increases, and they may display a distinct behavioral preference for the more intense of several signals (Kirkpatrick and Ryan, 1991; Ryan and Keddy- Hector, 1992). These generalizations are derived from laboratory and field studies of signaling in acoustic, vibrational and visual modalities, and they may represent a universal phenomenon (Greenfield, 2002). At a neuroethological level, increased responses to more intense signals may simply reflect that higher stimulus energy evokes an increased likelihood of messages from sensory neurons and a greater rate of action potentials in those messages (Ewert, 1980). From the perspective of sexual selection, females exhibiting such graded responses may obtain genetic or material benefits by virtue of mating with a male that can or does expend greater energy on his advertisements (Lande, 1981; Pomiankowski, 1988; Kokko et al., 2002). This fundamental response to signal intensity, however, can mask some critical details in the processing that females use to evaluate signals and how such evaluations might have shaped the evolution of male signaling efforts. Specifically, signal intensity normally fluctuates over different temporal scales, and it is not clear how females process and evaluate intensity given such fluctuations. Neurophysiologists have addressed one aspect of this problem through investigations of ‘integration time constants’ in perception. For example, acoustic species, particularly among insects and anurans, may integrate the sound energy that has arrived over a brief time interval (Tougaard, 1996; Tougaard, 1999; Ronacher et al., 2000; Gerhardt and Huber, 2002; Wyttenbach and Farris, 2004). This interval extends backward from the present time to a given moment in the past, and it is thus continually updated. The animal then processes a running integral of acoustic energy (sound amplitude summated over time) and can therefore detect and evaluate short pulses of sound and brief silent gaps, as well as other features, in longer broadcasts of song (e.g. Schiolten et al., 1981; Hennig et al., 2004). But depending on the parameters of the integration time constant, the animal may also fail to detect shorter pulses and briefer gaps. Despite the above studies on neuroethological function, relatively few behavioral studies have probed the manner in which time constants might influence the various processes of sexual selection. That is, we have very little information on the specific way in which females respond to the amplitude of male song. Do females simply assess overall mean amplitude levels and orient toward songs with the highest mean values, or are females unduly influenced by occasional maximum values that greatly exceed the mean? And, do the amplitude properties of male song show evidence of selection pressure imposed by the specific protocol of female assessment? We addressed the question of female evaluation of amplitude in male song in an acoustic moth, Achroia grisella Fabricius (lesser waxmoth; Lepidoptera: Pyralidae: Galleriinae). Male A. grisella attract females with a calling song that consists of a continuous train of ultrasonic pulses (Spangler et al., 1984). The males produce their song for 6–10 h each night until death while remaining stationary on the substrate and fanning their wings at 35–50 cycles per second (measured at 25°C). This activity causes a pair of small tymbal structures situated at the base of each forewing to resonate, once The Journal of Experimental Biology 212, 4091-4100 Published by The Company of Biologists 2009 doi:10.1242/jeb.035345 Evaluation of amplitude in male song: female waxmoths respond to fortissimo notes D. Limousin* and M. D. Greenfield Institut de recherche sur la biologie de l’insecte (IRBI), CNRS UMR 6035, Université François Rabelais de Tours, Parc de Grandmont, 37200 Tours, France *Author for correspondence (denis.limousin@etu.univ-tours.fr) Accepted 19 August 2009 SUMMARY Female evaluation of male signals in the context of sexual selection is often made on the basis of signal energy. Particularly in acoustic species, females may prefer male song that is broadcast at greater amplitude or power. However, song amplitude may be represented by various parameters, and the specific one(s) that are evaluated are not clear. We addressed this problem in an acoustic moth, Achroia grisella (Lepidoptera: Pyralidae), where males attract females with trains of paired ultrasonic pulses. Previous studies showed that females prefer songs that include pulse pairs that have greater mean peak amplitude and that are delivered with greater power (mean peak amplitudepulse-pair rate). Here, we report that given male songs of equal acoustic power, females prefer songs in which some pulses attain peak amplitudes that exceed the mean value and that this preference depends largely on the magnitude of amplitude fluctuation. We measured significant variation among males in their degree of amplitude fluctuation, and we note that males that broadcast with lower acoustic power typically show greater relative fluctuations and attain relatively higher amplitude maxima. We discuss the potential role of multiple integration time constants in female evaluation of mean song amplitude and amplitude maxima. We then consider the possibility that the variation observed in the male population is a response to female choice, but we also indicate that mechanical factors constraining song production may be responsible for such variation. Key words: acoustic power, fluctuating signal, integration time constant, signal evolution, song amplitude. THEJOURNALOFEXPERIMENTALBIOLOGY