The Journal of Experimental Biology © 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 4347-4355 doi:10.1242/jeb.113365 4347 ABSTRACT Sound is a widely available and vital cue in aquatic environments, yet most bioacoustic research has focused on marine vertebrates, leaving sound detection in invertebrates poorly understood. Cephalopods are an ecologically key taxon that likely use sound and may be impacted by increasing anthropogenic ocean noise, but little is known regarding their behavioral responses or adaptations to sound stimuli. These experiments identify the acoustic range and levels that elicit a wide range of secondary defense behaviors such as inking, jetting and rapid coloration change. Secondarily, it was found that cuttlefish habituate to certain sound stimuli. The present study examined the behavioral responses of 22 cuttlefish (Sepia officinalis) to pure-tone pips ranging from 80 to 1000 Hz with sound pressure levels of 85–188 dB re. 1 μPa rms and particle accelerations of 0–17.1 m s −2 . Cuttlefish escape responses (inking, jetting) were observed between frequencies of 80 and 300 Hz and at sound levels above 140 dB re. 1 μPa rms and 0.01 m s −2 (0.74 m s −2 for inking responses). Body patterning changes and fin movements were observed at all frequencies and sound levels. Response intensity was dependent upon stimulus amplitude and frequency, suggesting that cuttlefish also possess loudness perception with a maximum sensitivity around 150 Hz. Cuttlefish habituated to repeated 200 Hz tone pips, at two sound intensities. Total response inhibition was not reached, however, and a basal response remained present in most animals. The graded responses provide a loudness sensitivity curve and suggest an ecological function for sound use in cephalopods. KEY WORDS: Bioacoustics, Cephalopod, Hearing, Noise, Loudness, Invertebrate, Ear, Statocyst, Lateral line INTRODUCTION Sound in aquatic environments is a widely available cue that many marine vertebrates use during vital biological activities such as foraging, predator detection, mate attraction and habitat selection (Webster et al., 1992; Fay and Popper, 1998; Au et al., 2000). Consequently, for vertebrates, sound detection is considered a primary sensory modality and an important component of vital intraspecific interactions and a key way to detect the surrounding environment. The ability of marine invertebrates to detect and potentially use sound is far less understood (Budelmann, 1992a; Budelmann, 1992b; Mooney et al., 2012). This is somewhat surprising given their relative abundance and central role in many marine ecosystems. RESEARCH ARTICLE 1 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. 2 Experimental Zoology Group, Wageningen University, De Elst 1, 6708WD Wageningen, The Netherlands. 3 Program in Sensory Physiology and Behavior, Marine Biological Laboratory, Woods Hole, MA, 02543, USA. *Author for correspondence (amooney@whoi.edu) Received 1 September 2014; Accepted 22 October 2014 Yet, a growing body of literature suggests that marine invertebrates respond to sound in a variety of ways. For example, coral reef invertebrates (crabs and coral larvae) may swim toward or away from reef sounds, with the actual direction being taxon specific (Vermeij et al., 2010; Simpson et al., 2011). Reef sounds from certain habitats can generate settlement behaviors and increased rates of metamorphosis (Stanley et al., 2010; Stanley et al., 2012). Perhaps not surprisingly, variations in the frequencies and levels of these sounds can affect whether the behavior is induced (Simpson et al., 2011; Stanley et al., 2011). However, thresholds have rarely been established and we still know little regarding the frequencies to which most invertebrates respond. Furthermore, it is vital to quantify acoustic particle motion, a stimulus often overlooked. Both sound pressure and acoustic particle motion are generated by sound sources, but it is particle motion [i.e. the back- and-forth hydrodynamic flow from the motion of the sound emitter (Gade, 1982; Au and Hastings, 2009)] that is the likely stimulus for most marine animals without compressible air cavities (Mann et al., 2007; Mooney et al., 2010; Popper and Fay, 2011). Despite a burgeoning literature, there is a poor understanding of the frequencies and levels of sounds that generate functional behavioral responses in invertebrates. Cephalopods offer a unique means to quantify the frequency range and sound levels that generate behavioral responses for several reasons. First, the potential behavioral responses of several species, such as the common cuttlefish, Sepia officinalis Linnaeus 1758, are both dynamic and well described (Hanlon and Messenger, 1996). Previous behavioral studies have shown that these cuttlefish exhibit a range of responses to sensory stimuli, including changes in body patterning, locomotor activity, jetting and inking events (Hanlon and Messenger, 1996). Second, these behavioral responses show a gradation in intensity, from primary defense responses (usually crypsis or camouflaging against the background), to secondary defenses such as deimatic behaviors used to deter the potential predator, and ultimately flight responses involving jetting and inking (Hanlon and Messenger, 1988; Langridge et al., 2007; Langridge, 2009; Staudinger et al., 2011). A similar gradation in response intensity may be generated by acoustic stimuli (Fewtrell and McCauley, 2012). Finally, many cephalopods occupy central positions in food chains; thus, understanding their sensory ecology is required to accurately determine relationships between this taxon and other marine species, and could provide indications on how other invertebrates may use sound. The statocyst is generally considered the primary sound detection organ in cephalopods (Budelmann, 1990; Budelmann, 1992a), although peripheral hair cells may play a role in detecting local water movements (Bleckmann et al., 1991; Coombs et al., 1992). With regard to acoustic stimuli, the statocyst likely acts as an accelerometer in response to the vibratory particle motion component of sound (Budelmann, 1990; Packard et al., 1990; Mooney et al., 2010). Besides the hair cells in the statocysts, Graded behavioral responses and habituation to sound in the common cuttlefish Sepia officinalis Julia E. Samson 1,2 , T. Aran Mooney 1,3, *, Sander W. S. Gussekloo 2 and Roger T. Hanlon 3