Hornworm counterattacks: defensive strikes and sound production in response to invertebrate attackers

Hornworm counterattacks: defensive strikes and sound production in response to invertebrate... Abstract Caterpillars (i.e. lepidopteran larvae) have evolved multiple defences against predators, with some large caterpillars showing aggressive defences (e.g. strikes and/or sound production). Although such behaviours can startle or warn vertebrate predators, defences against invertebrates remain unclear. We investigated the behavioural responses of the hornworm Langia zenzeroides (Lepidoptera: Sphingidae) against the invertebrate attacker Calosoma maximowiczi (Coleoptera: Carabidae). Fifth (last) instars of L. zenzeroides exhibited a striking response, in which the larva rapidly bent its head and thorax towards the body part stimulated by C. maximowiczi attacks. Strikes were also accompanied by opening of the mandibles, followed by sound production or regurgitation. In some cases, L. zenzeroides larvae caught the legs of C. maximowiczi and threw the beetles using their mandibles. Such counterattacks completely defended against attackers. The sounds that L. zenzeroides generated (pulse durations, 82–314 ms; dominant frequencies, 5.0–8.7 kHz; sound pressure level, 44.0–56.9 dB SPL) were produced by forcing air through the eighth pair of abdominal spiracles. Our results indicate that hornworm larvae are able to deter predacious invertebrates using multiple defences. INTRODUCTION Prey animals have evolved defensive behaviours against predators (Edmunds, 1974; Ruxton, Sherratt & Speed, 2004). During attack, some animals retaliate by biting, scratching, stinging, or discharging toxic chemicals that can damage the predators (Edmunds, 1974; Schmidt, 1990; Mukherjee & Heithaus, 2013). If the predators are injured by aggressive defences, they may give up their attack (cf. Sugiura & Yamazaki, 2014). Aggressive animal species that can injure predators are more likely to survive than are species that cannot. Thus, various types of aggressive defences have evolved in several animal taxa (Edmunds, 1974; Schmidt, 1990). Aggressive animal species may also warn their predators using their body colour, sounds, defensive devices and behaviours, thus forcing predators to avoid the prey (Schmidt, 1990). Caterpillars, including lepidopteran larvae, have multiple forms of defensive behaviour in response to predators (Greeney, Dyer & Smilanich, 2012). Larvae of the family Sphingidae (i.e. hornworms or hawkmoths) are relatively large and display aggressive defences against attackers (Walters et al., 2001; Brown, Boettner & Yack, 2007; Bura, Kawahara & Yack, 2016). For example, late instars of a hornworm species have a ‘defensive strike’ that includes rapid bending that accurately propels the head towards the abdominal site stimulated by attackers (Walters et al., 2001; Simon & Trimmer, 2009; van Griethuijsen, Banks & Trimmer, 2013). Multiple strikes (thrashing) can decrease the success of birds biting caterpillars (Walters et al., 2001). Striking behaviour is also accompanied by opening of the mandibles and occasional regurgitation (Walters et al., 2001); regurgitants can function as chemical defences against predators (Grant, 2006; Brown et al., 2007; Greeney et al., 2012). Furthermore, late instars of several hornworm species produce sounds in response to physical stimuli (Brown et al., 2007; Bura et al., 2011, 2012, 2016), which can deter vertebrate predators, such as birds (Brown et al., 2007; Bura et al., 2011; Dookie et al., 2017). Vertebrates, including birds, are important predators of large caterpillars in natural conditions (Stewart, 1975; Remmel, Davison & Tammaru, 2011). Therefore, previous studies have focused on vertebrate predators as important agents for providing selective pressures on the evolution of aggressive defences in caterpillars belonging to the superfamily Bombycoidea (including the families Sphingidae and Saturniidae; Brown et al., 2007; Bura et al., 2011, 2016). Arthropod predators can also pose strong predation pressures on hornworm larvae (Madden & Chamberlin, 1945; Lawson, 1959). Although hornworms frequently encounter invertebrate predators and parasitoids on host plants and on the ground (cf. Madden & Chamberlin, 1945; Bellotti, Arias & Guzman, 1992), it is unclear whether hornworms show aggressive defences (i.e. striking behaviour, regurgitation and/or sound production) against invertebrate predators. Could hornworm defences be effective against invertebrate attackers? Defensive strikes and regurgitation can function as behavioural and chemical defences, respectively, against attackers (Walters et al., 2001; Brown et al., 2007). Some invertebrate predators may respond to sounds and/or substrate-borne vibrations produced by hornworm larvae (Bura et al., 2016), although many invertebrate predators lack tympanal ears that are sensitive to sounds with broad frequencies (Yager, 1999; Yack, 2004). Determining the defences of hornworms in response to invertebrate attackers would improve our understanding of how defensive behaviours have evolved in caterpillars. To determine the function and mechanism of hornworm defences against invertebrate attackers, we investigated such behaviour in the hornworm Langia zenzeroides Moore (Lepidoptera: Sphingidae) in response to attacks by the caterpillar-feeding beetle Calosoma maximowiczi Morawitz (Coleoptera: Carabidae). Langia zenzeroides is the largest hornworm in Japan (body length of last instars: 100–130 mm; Yasuda, 2010; Kishida, 2011) and produces sounds in the larval, prepupal and adult stages (Inoue et al., 1982). In addition, the phylogenetic position of L. zenzeroides is unique in the plesiomorphic condition of the two subfamilies (i.e. Smerinthinae and Sphinginae; Kawahara et al., 2009). Therefore, L. zenzeroides is a model hornworm for investigating how aggressive defences have evolved in the family Sphingidae. The adults of the carabid genus Calosoma are well known as caterpillar hunters (Bruschi, 2013; Toussaint & Gillett, 2017), providing an appropriate predator for investigating the defensive behaviour of caterpillars (Sugiura & Yamazaki, 2014; Sugiura, 2016). Thus, we observed the responses of L. zenzeroides larvae to C. maximowiczi attacks in laboratory conditions. In addition, we characterized and clarified the mechanisms of sound production by the hornworm. Finally, we discuss the effectiveness of aggressive defences in hornworm caterpillars. MATERIAL AND METHODS Study species In this study, L. zenzeroides larvae were reared from eggs obtained from two female adults that were captured in Takarazuka, Hyogo Prefecture, central Japan (34°51′N, 134°18′E, 130 m above sea level) in early April 2015. Langia zenzeroides grows through five larval instars before pupating (Yasuda, 2010). The instar was determined based on the maximal width of the head capsule (first instar, 1.4–1.5 mm; second instar, 1.9–2.2 mm; third instar, 2.9–3.1 mm; fourth instar, 4.4–4.6 mm; fifth instar, 6.1–6.6 mm). Larvae were reared on leaves of Cerasus × yedoensis (Matsum.) A.V. Vassil. and Cerasus speciosa (Koidz.) H. Ohba (Rosaceae) in laboratory conditions (25 °C, 16 h–8 h light–dark cycle). Fifth instars of L. zenzeroides produced sounds in response to physical stimuli (see Supporting information, Movie S1). In laboratory experiments, we used larvae that were randomly chosen from among 37 fifth instars of L. zenzeroides (body length, mean ± SEM, 100.4 ± 2.1 mm). Several larvae were used in different experiments. Before experimentation, the body length of each hornworm was measured to the nearest 0.1 mm using slide callipers while it was resting on a twig. Calosoma maximowiczi adults attack multiple caterpillar species on the ground and in vegetation (Sugiura & Yamazaki, 2014; Sugiura, 2016). As found in some predatory animals (e.g. Sugiura et al., 2011; Ohba & Tatsuta, 2016), C. maximowiczi adults were able to attack prey larger than themselves (Sugiura, 2016). For the following experiments, all of the adults of C. maximowiczi were collected from a secondary forest in Kobe, Hyogo (34°42′N, 134°11′E, 60–170 m above sea level), in early May 2015. Although we have not observed C. maximowiczi attacking hornworms in field conditions, the habitat and active season overlap between C. maximowiczi adults and L. zenzeroides larvae. Therefore, L. zenzeroides larvae might encounter C. maximowiczi on host plants or on the ground. Twenty-five C. maximowiczi adults were used in this study, and the body length of each adult was measured to the nearest 0.1 mm using slide callipers. Behavioural experiment To elucidate defensive behaviour against invertebrate attackers, we investigated the response of L. zenzeroides larvae to bites and other physical contact by the invertebrate attacker C. maximowiczi. The experiment was performed during the day in May 2015 in a well-lit laboratory (25 ± 1 °C). Given that C. maximowiczi adults and L. zenzeroides larvae naturally forage on twigs and trunks, a carabid adult and a hornworm were placed on bamboo (width, 7 mm; height, 15 mm; Fig. 1; cf. Sugiura 2016). The bamboo was looped (length, 700 mm; diameter, 200 mm; Fig. 1) such that hornworms could encounter carabids in all trials. The looped bamboo was also surrounded by a plastic circular cylinder (diameter, 220 mm; height, 120 mm; Fig. 1). We used 25 L. zenzeroides larvae (body length, mean ± SEM, 105.4 ± 1.9 mm) and 25 C. maximowiczi adults in the experiment. Figure 1. View largeDownload slide The arena used in our experiments. (A) A Langia zenzeroides larva and a Calosoma maximowiczi adult placed on bamboo material (Sugiura, 2016). (B) Overhead view of the arena (looped bamboo material) surrounded by a plastic circular cylinder. Scale bars: 15 mm. Figure 1. View largeDownload slide The arena used in our experiments. (A) A Langia zenzeroides larva and a Calosoma maximowiczi adult placed on bamboo material (Sugiura, 2016). (B) Overhead view of the arena (looped bamboo material) surrounded by a plastic circular cylinder. Scale bars: 15 mm. Immediately before the experiment, the activity of C. maximowiczi was assessed using the prey caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) (sixth instar, 22.0–35.0 mm in length). We used forceps to move prey caterpillars in front of C. maximowiczi. When C. maximowiczi attacked (bit) prey caterpillars, we considered them to be active. Eleven C. maximowiczi (four males and seven females; body length, mean ± SEM, 25.7 ± 0.5 mm) attacked prey caterpillars (hereafter, ‘active beetles’), and 14 adults did not (hereafter, ‘inactive beetles’; six males and eight females; body length, mean ± SEM, 26.7 ± 0.4 mm). The sex ratios and body lengths were not significantly different between active and inactive beetles (sex ratio, Fisher’s exact test, P = 1.0; body length, t-test, t = −1.4443, P = 0.16). All active beetles bit prey with their mandibles. Although inactive adults did not bite prey, their body parts frequently touched the prey bodies. Thus, we used active and inactive C. maximowiczi adults to investigate the defensive responses of L. zenzeroides larvae to bites and other physical touches (without bites) by C. maximowiczi, respectively. First, we placed a hornworm on bamboo. When the hornworm rested on the bamboo, a carabid adult was placed on the far end of the bamboo (Fig. 1). During a 10 min period, recordings were obtained for whether a carabid beetle used mandibles to attack L. zenzeroides, how many times the carabid contacted the larva, how the larva responded to the contacts (i.e. strike, sound production and/or regurgitation) and how the carabid responded to L. zenzeroides defences (i.e. retreat or continue to attack). We also recorded the body part (head, thorax, abdomen A1–3, A4–7 or A8–10) touched or bitten by the carabid and the responses in each L. zenzeroides larva. After an L. zenzeroides larva successfully repelled a beetle, we continued to watch for further attacks by C. maximowiczi within the 10 min period (cf. Sugiura, 2016). Defensive behaviour was recorded using the movie function of a digital camera (iPhone 6 plus; Apple) at 240 frames s−1. Sound and video recordings The sounds produced by randomly chosen larvae (body length, mean ± SEM, 89.0 ± 4.8 mm; N = 9) were recorded. Two of nine larvae were also used in the behavioural experiment. The recordings were made before the behavioural experiment. Each larva was placed on a cut twig, which was perpendicular to the ground, so that the larva hung its head down from the posterior prolegs. Larvae were stimulated from both sides of the abdomen (A8–A10) using forceps. During these trials, the larvae produced sounds, which were recorded using a 1/4-inch condenser microphone (type 4939; Brüel & Kjær, Nærum, Denmark) in a soundproof room. Sound signals were amplified (type 2670 and 2690, with a 0.02–100 kHz bandpass filter; Brüel and Kjær) and digitized using an analog converter with a 0.7 Hz high-pass filter (PULSE type 3560-B; Brüel & Kjær) at a sampling rate of 65.5 kHz (24 bits). The digitized sounds were analysed using PULSE Labshop software (version 15.1.0; Brüel & Kjær). The microphone was held at a distance of 20 mm from the dorsal surface of the larval abdomen, and power spectra were computed in the PULSE Reflex software (version 18.1.1; Brüel & Kjær) using a Hanning window (fast Fourier transform, line, 400; frequency resolution, 128 Hz; frequency span, 51.2 kHz). The spectral characteristics were analysed with a high-pass filter at 500 Hz. The average and individual spectra of sounds recorded from nine larvae were calculated after the conversion of logarithmic decibels into linear micropascals. Sound levels were determined as the decibel peak equivalent sound pressure level (dB peSPL; 0 dB = 20 μPa rms), referring to signals from a sound level calibrator (type 4230, 94 dB SPL at 1 kHz; Brüel & Kjær). A pulse is defined as a group of uninterrupted waves or elements (Bura et al., 2011). Temporal characters of pulse durations and dominant frequencies were measured from the recordings of nine larvae. Similar methods have been used in previous studies (cf. Takanashi et al., 2010; Bura et al., 2011; Tsubaki et al., 2014). The strike behaviour of randomly chosen larvae (body length, mean ± SEM, 87.2 ± 5.1 mm; N = 7) was filmed using a high-speed camera (FASTCAM Mini UX50; Photron, Japan) with a Zoom-Nikkor lens (35–70 mm, f/3.3–4.5; Nikon, Japan) at 500 frames s−1. We measured the time required from the start of movement to striking the head against the forceps. When multiple strikes occurred, we measured the duration of the first strike. Sounds were recorded at the same time using the type 4939 microphone placed 20 mm away from the dorsal abdomen. Signals from the microphone were transmitted as described above, and the recorded videos were combined with the audio (FASTCAM Analysis, version 1.2.1.1; Photron, Japan) and analysed temporally. Sound production mechanism To clarify the mechanisms leading to sound production in L. zenzeroides larvae, we tested ‘clicking’ by the mandibles (Brown et al., 2007; Bura et al., 2012), ‘whistling’ through the abdominal spiracles (Bura et al., 2011) and ‘vocalizing’ through the oral cavity (Bura et al., 2016). First, larval mandibles were observed using the digital camera (iPhone 6 plus) during sound production, and the movement of seven larvae (body length, mean ± SEM, 89.9 ± 5.2 mm) was played back using QuickTime Player version 10.4 (Apple, Inc.). Second, ten larvae (body length, mean ± SEM, 97.9 ± 3.0 mm) were individually submerged in a plastic container (diameter, 155 mm; height, 60 mm) filled with water (300 mL) and pinched with forceps. Behaviour was filmed using a video camera (Handycam, HDR-CX630; Sony, Japan), and the QuickTime Player was used to check whether air bubbles emerged from abdominal spiracles, the mouth or other body locations. Statistical analysis Fisher’s exact tests were performed to compare the frequency of strike behaviour, sound production and regurgitation between different responses (i.e. responses to active or inactive beetles). A generalized linear model (GLM) with Poisson error distribution and log link was used to clarify the effects of attacker types (i.e. active or inactive beetles) on the number of contacts with L. zenzeroides larvae. The number of physical contacts between beetle attackers and L. zenzeroides larvae was used as a response variable, whereas active and inactive beetles were treated as fixed factors. A quasi-Poisson error distribution was used when the residual deviance was larger than the residual degrees of freedom (i.e. overdispersion; Crawley, 2005). All the analyses were performed using R version 3.2.2 (R Development Core Team, 2015). RESULTS Defensive behaviour in response to carabid beetles The number of physical contacts with L. zenzeroides larvae ranged from 12 to 38 and from seven to 48 in active and inactive beetles, respectively, which was not significantly different (GLM, taking into account overdispersion, t = −1.143, P = 0.25). Heads, thoraxes and/or abdomens of L. zenzeroides larvae were touched or bitten by the beetles (see Supporting information, Table S1). All the active beetles attacked L. zenzeroides larvae that were 3.4–4.6 times longer, and all active beetles used their mandibles to bite the heads or abdomens of L. zenzeroides larvae at least once (Fig. 2A). All L. zenzeroides larvae (N = 11) whose heads, thoraxes or abdomens were stimulated by carabids exhibited striking responses to active beetles by bending their heads towards the location that was bitten or contacted (Fig. 2B, Table 1; see Supporting information, Table S2). Langia zenzeroides larvae also exhibited multiple strikes (thrashing) while being repeatedly attacked by carabid beetles (see Supporting information, Movie S2). All the larvae (N = 11) exhibiting strike behaviour simultaneously produced sounds that were audible to the human ear (Table 1); sound production was confirmed when heads, thoraxes and/or abdomens of L. zenzeroides larvae were attacked by the beetles (see Supporting information, Table S3). Regurgitation was also accompanied by strike behaviour and sound production in 54.5% (N = 6/11) of the larvae that were bitten by beetles (Table 1). Leaf fragments were included in the regurgitant. Two larvae (18.2%) caught the legs of beetles using their mandibles and threw them. Additionally, one of the two larvae removed the distal portion of the beetle’s right hindleg (tibia and tarsus) by biting (Fig. 3; see Supporting information, Movie S2). No beetles killed L. zenzeroides larvae, and two active beetles retreated in response to L. zenzeroides strikes and bites. Therefore, such counterattacks could effectively defend L. zenzeroides against carabid attacks. Table 1. Defensive behaviours of Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi   Percentage of L. zenzeroides larvae (N)*  Behavioural responses  Bites and/or contacts by active beetle  Physical contacts by inactive beetle  Strike, sound and regurgitation  54.5 (6)  7.1 (1)  Strike and sound  45.5 (5)  42.9 (6)  Strike and regurgitation  0.0 (0)  0.0 (0)  Strike (only)  0.0 (0)  35.7 (5)  Sound and regurgitation  0.0 (0)  0.0 (0)  Sound (only)  0.0 (0)  0.0 (0)  Regurgitation (only)  0.0 (0)  0.0 (0)  None  0.0 (0)  14.3 (2)  Total  100.0 (11)  100.0 (14)    Percentage of L. zenzeroides larvae (N)*  Behavioural responses  Bites and/or contacts by active beetle  Physical contacts by inactive beetle  Strike, sound and regurgitation  54.5 (6)  7.1 (1)  Strike and sound  45.5 (5)  42.9 (6)  Strike and regurgitation  0.0 (0)  0.0 (0)  Strike (only)  0.0 (0)  35.7 (5)  Sound and regurgitation  0.0 (0)  0.0 (0)  Sound (only)  0.0 (0)  0.0 (0)  Regurgitation (only)  0.0 (0)  0.0 (0)  None  0.0 (0)  14.3 (2)  Total  100.0 (11)  100.0 (14)  *Values in parentheses indicate the numbers of L. zenzeroides larvae. View Large Figure 2. View largeDownload slide The hornworm Langia zenzeroides and its potential attacker Calosoma maximowiczi. (A) An adult C. maximowiczi biting the head of an L. zenzeroides larva. (B) An L. zenzeroides larva striking an adult C. maximowiczi. (C) A fifth instar of L. zenzeroides. One and eight pairs of spiracles are paced on the thorax (T1) and abdomen (A1–A8), respectively. The arrow indicates the spiracle on the eighth abdominal segment (A8). Scale bars: 10 mm. Figure 2. View largeDownload slide The hornworm Langia zenzeroides and its potential attacker Calosoma maximowiczi. (A) An adult C. maximowiczi biting the head of an L. zenzeroides larva. (B) An L. zenzeroides larva striking an adult C. maximowiczi. (C) A fifth instar of L. zenzeroides. One and eight pairs of spiracles are paced on the thorax (T1) and abdomen (A1–A8), respectively. The arrow indicates the spiracle on the eighth abdominal segment (A8). Scale bars: 10 mm. Figure 3. View large Download slide Sequential images of Langia zenzeroides behaviour in response to Calosoma maximowiczi (see Supporting information, Movie S2). The strike (150 ms), bite (450 ms), throw (600–750 ms) and regurgitation times (600–800 ms) were observed. The arrows indicate the beetle leg removed by the larva. Scale bar: 15 mm. Figure 3. View large Download slide Sequential images of Langia zenzeroides behaviour in response to Calosoma maximowiczi (see Supporting information, Movie S2). The strike (150 ms), bite (450 ms), throw (600–750 ms) and regurgitation times (600–800 ms) were observed. The arrows indicate the beetle leg removed by the larva. Scale bar: 15 mm. All inactive beetles touched L. zenzeroides larvae but not to bite them. Physical contacts by inactive beetles stimulated 85.7% of the larvae (N = 12/14) to evoke striking responses (Table 1). Sound production accompanied striking behaviour in 58.3% (N = 7/12) of the larvae (Table 1), and no responses were observed in 14.3% of the larvae touched by inactive beetles (Table 1). Only one larva regurgitated, when its posterior abdomen was merely touched by an inactive beetle (Table 1; see Supporting information, Table S4). The frequency of strike behaviour in larvae touched by inactive beetles was not significantly different from that of larvae bitten by active beetles (Fisher’s exact test; sound production, P = 0.49). However, larvae bitten by active beetles produced sound and regurgitated more frequently than larvae contacted by inactive beetles (Fisher’s exact test; sound production, P = 0.0078; regurgitation, P = 0.0213). Strike behaviour, sound characteristics and mechanism of sound production Langia zenzeroides larvae performed a striking response to abdominal pinching by forceps, in which they rapidly bent their heads and thoraxes towards the pinched abdomen (see Supporting information, Movie S1). The duration of the behaviour (from start of bending to striking) ranged from 142 to 230 ms (mean ± SEM, 180.6 ± 13.0 ms; N = 7; see Supporting information, Movie S3). The larvae produced a single sound pulse in response to artificial stimuli (see Supporting information, Movie S3, Audio S1). The pulse duration ranged from 82 to 314 ms (mean ± SEM, 166.7 ± 21.1 ms; N = 9; Fig. 4A), and spectral analyses revealed that the dominant frequency ranged from 5.0 to 8.7 kHz (mean ± SEM, 5.9 ± 0.4 kHz; N = 9; Fig. 4B), and the maximal sound pressure ranged from 44.0 to 56.9 dB SPL (mean, 49.8 dB SPL, N = 9; Fig. 4B). Larval mandibles (N = 7) remained open during sound production, suggesting that L. zenzeroides does not produce sound with their mandibles. When L. zenzeroides larvae were submerged in water, 30% (N = 3/10) produced air bubbles from a pair of spiracles on the eighth abdominal segment with sounds that were audible to the human ear (Fig. 2C; see Supporting information, Movie S4), indicating that L. zenzeroides larvae ‘whistle’ through a pair of eighth abdominal spiracles. The lack of sound production in some larvae was probably the result of being submerged in water. Figure 4. View large Download slide Characteristics of the sounds produced by Langia zenzeroides larvae. (A) An oscillogram of the sound pulse produced by a larva (see Supporting information, Audio S1). (B) Power spectra of larval pulses. The black and red lines are derived from the pulse of the larva (shown in the oscillogram) and the pulses of nine larvae (i.e. means of N = 9), respectively. Figure 4. View large Download slide Characteristics of the sounds produced by Langia zenzeroides larvae. (A) An oscillogram of the sound pulse produced by a larva (see Supporting information, Audio S1). (B) Power spectra of larval pulses. The black and red lines are derived from the pulse of the larva (shown in the oscillogram) and the pulses of nine larvae (i.e. means of N = 9), respectively. DISCUSSION Aggressive defences that can damage or kill attackers have been reported in some animal species (Edmunds, 1974; Schmidt, 1990; Mukherjee & Heithaus, 2013). Lepidopteran larvae show various types of aggressive defences, including defensive strikes, sound production and regurgitation (Walters et al., 2001; Brown et al., 2007; Greeney et al., 2012; Bura et al., 2016). However, only a few studies have focused on which stimuli cause aggressive defences and how the aggressive defences might be effective (Walters et al., 2001; Dookie et al., 2017). In the present study, we showed that C. maximowiczi attacks caused L. zenzeroides larvae to exhibit striking behaviour and/or sound production (Table 1). Beetle bites also frequently induced regurgitation (Table 1). Therefore, such aggressive defences could effectively protect L. zenzeroides larvae from beetle attack, increasing larval survivorship. Counterattacks The number of physical contacts with L. zenzeroides larvae did not differ between active and inactive beetles. In addition, the frequency of strike behaviour in larvae touched by inactive beetles was not different from that of larvae bitten by active beetles (Table 1). These results suggest that L. zenzeroides larvae exhibit strike behaviour in response to almost all physical contact with beetles (including biting). Although many hornworm species exhibit strike behaviour (Edmunds, 1974; Wagner, 2005), detailed assessments of such behaviours have been performed only in Manduca sexta (L.) (Walters et al., 2001; Simon & Trimmer, 2009; van Griethuijsen et al., 2013). Walters et al. (2001) showed that thrashing (repeated strikes) of M. sexta larvae decreased the success of avian predators. Although the strike behaviour of M. sexta larvae is thought to dislodge smaller predators (Walters et al., 2001), hornworm defences against predacious invertebrates have remained unexplored. Langia zenzeroides larvae counterattacked C. maximowiczi by striking (Fig. 3; see Supporting information, Movie S2), suggesting that hornworm strikes function as a behavioural defence against predacious invertebrates. In addition, L. zenzeroides larvae threw C. maximowiczi when legs were lodged in their mandibles, and it is surprising that the soft-bodied caterpillar injured the hard-bodied beetle (Fig. 3; see Supporting information, Movie S2). Although some hornworm species nip at their attackers (Wagner, 2005), such an aggressive defence has rarely been reported. Langia zenzeroides larvae bitten by active beetles produced sound and regurgitated more frequently than larvae contacted by inactive beetles, although larvae contacted by inactive beetles exhibited strike behaviour as frequently as those bitten by active beetles (Table 1). Therefore, beetle contact stimuli caused L. zenzeroides larvae to exhibit a single defence (strike behaviour), whereas intense stimuli (i.e. beetle bites) induced L. zenzeroides larvae to exhibit multiple defences (strikes, sound production and regurgitation). Leaf fragments included in the larval regurgitants suggest that L. zenzeroides larvae regurgitated their gut contents; such regurgitants are considered chemical defences against predators (Grant, 2006; Brown et al., 2007; Greeney et al., 2012). Collectively, the multiple defences of L. zenzeroides larvae routinely repelled carabid attackers (Fig. 3; see Supporting information, Movie S2), although the costs of multiple defences are higher than those of a single defence (strike behaviour). Given that only one insect species (i.e. C. maximowiczi) was used as a model predator in this study, the defensive responses of L. zenzeroides larvae against other types of predators remain unclear. Various enemies might attack L. zenzeroides larvae in field conditions, although no enemies have been recorded. Therefore, further experiments are needed to clarify the effectiveness of L. zenzeroides defences against invertebrate predators and parasitoids. Sound production and its mechanism Sound production has been reported in all three subfamilies of Sphingidae (i.e. Macroglossinae, Sphinginae and Smerinthinae; Bura et al., 2016). In two subfamilies, Macroglossinae and Sphinginae, clicking of the mandibles produces sound. Vocalizing through the oral cavity occurs only in Macroglossinae, and whistling through the abdominal spiracles occurs in Smerinthinae (Bura et al., 2016). Our data revealed that L. zenzeroides larvae produce sound by forcing air through a pair of eighth abdominal spiracles (Fig. 2C; see Supporting information, Movie S4). A similar mechanism has been reported in the smerinthine caterpillar Amorpha juglandis (Smith), although the dominant frequencies and sound pressure levels of L. zenzeroides larvae were marginally lower than those of A. juglandis larvae (Bura et al., 2011). Kawahara et al. (2009) determined that the placement of L. zenzeroides within the phylogeny is unique in that it illustrates the plesiomorphic condition of the Smerinthinae and Sphinginae. Given that sound production is known in only two smerinthines (Bura et al., 2016), and because Langia and Amorpha are not closely related, it appears that these similar mechanisms might have arisen independently. The sounds produced by hornworm larvae can frighten vertebrate predators (startle displays; Brown et al., 2007; Bura et al., 2016) or warn them (acoustic aposematism; Bura et al., 2011, 2016; Dookie et al., 2017); however, the defensive function against invertebrates remains unclear. Some coleopteran insects, including cicindelines and scarabaeids, have tympanal ears that are sensitive to sounds with broad frequencies (Yager, 1999; Yack, 2004). Whether the carabid beetle C. maximowiczi has tympanal ears to detect sounds made by L. zenzeroides remains unclear. However, C. maximowiczi might be able to detect substrate-borne vibrations produced by L. zenzeroides (cf. Autrum & Schneider, 1948). In addition, many L. zenzeroides larvae that produced sounds simultaneously exhibited strike behaviour and/or regurgitation (Table 1). Given that defensive strikes and regurgitants can damage predacious invertebrates, L. zenzeroides sounds might function as a warning against invertebrate attackers. However, our results did not fully clarify the defensive functions of L. zenzeroides sounds against invertebrate attackers, and the responses of vertebrate attackers to L. zenzeroides sounds remain unclear. Additional experiments are required to determine the defensive functions of L. zenzeroides sounds. CONCLUSION Although the strike behaviours of hornworm caterpillars startle vertebrate predators (Walters et al., 2001), the present study indicated that L. zenzeroides strikes deter predacious beetles. This is the first demonstration of the defensive functions of hornworm strikes against predacious invertebrates. Multiple defences, including strikes, throwing, regurgitation and sound production, could effectively discourage invertebrate attackers. ACKNOWLEDGEMENTS We thank T. Ohshio and I. Hanatani for providing L. zenzeroides adults and assistance with the rearing of caterpillars, respectively. We also thank the Editor and several reviewers for their valuable comments on our manuscript. This research was partly supported by Grants-in-Aid for Scientific Research (26450065 and 17K08158). Our experiments were conducted in accordance with the Kobe University Animal Experimentation Regulations (Kobe University’s Animal Care and Use Committee, H27). The experiments also complied with the current laws of Japan. The authors declare there are no competing financial interests. S.S. and T.T. conceived and designed experiments. S.S. collected and reared insects and performed behavioural experiments. S.S. and T.T. carried out sound recording and analyses and analysed the data. S.S. wrote the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Numbers of contacts between Langia zenzeroides larvae and Calosoma maximowiczi adults. Table S2. Percentage of strike behaviour in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Table S3. Percentage of sound production in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Table S4. Percentage of regurgitation in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Movie S1. Strike behaviour of Langia zenzeroides larvae in response to artificial stimuli (i.e. pinched by forceps). Movie S2. Defensive behaviour of Langia zenzeroides larvae in response to physical contacts or attacks by Calosoma maximowiczi. Movie S3. Slow-motion video of strike and sound production by Langia zenzeroides in response to an artificial stimulus. The vertical axis indicates the timing of the left video of the strike, in the right figure, without a high-pass sound filter applied to the oscillogram. Movie S4. Mechanism of sound production in Langia zenzeroides. The submerged larva produced air bubbles from a pair of eighth abdominal spiracles. Sounds were detectable by the human ear. Audio S1. Sounds of a Langia zenzeroides larva. SHARED DATA Data available from the Figshare Digital Repository (Sugiura & Takanashi, 2018). REFERENCES Autrum H, Schneider W. 1948. Vergleichende Untersuchungen über den Erschütterungssinn der Insekten (in German). Zeitschrift für Vergleichende Physiologie  31: 77– 88. Google Scholar CrossRef Search ADS PubMed  Bellotti AC, Arias B, Guzman OL. 1992. Biological control of the cassava hornworm Erinnyis ello (Lepidoptera: Sphingidae). Florida Entomologist  75: 506– 515. Google Scholar CrossRef Search ADS   Brown SG, Boettner GH, Yack JE. 2007. Clicking caterpillars: acoustic aposematism in Antheraea polyphemus and other Bombycoidea. The Journal of Experimental Biology  210: 993– 1005. Google Scholar CrossRef Search ADS PubMed  Bruschi S. 2013. Calosoma of the world . Bologna: Natura Edizioni Scientifiche. 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Hornworm counterattacks: defensive strikes and sound production in response to invertebrate attackers

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© 2018 The Linnean Society of London, Biological Journal of the Linnean Society
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0024-4066
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Abstract

Abstract Caterpillars (i.e. lepidopteran larvae) have evolved multiple defences against predators, with some large caterpillars showing aggressive defences (e.g. strikes and/or sound production). Although such behaviours can startle or warn vertebrate predators, defences against invertebrates remain unclear. We investigated the behavioural responses of the hornworm Langia zenzeroides (Lepidoptera: Sphingidae) against the invertebrate attacker Calosoma maximowiczi (Coleoptera: Carabidae). Fifth (last) instars of L. zenzeroides exhibited a striking response, in which the larva rapidly bent its head and thorax towards the body part stimulated by C. maximowiczi attacks. Strikes were also accompanied by opening of the mandibles, followed by sound production or regurgitation. In some cases, L. zenzeroides larvae caught the legs of C. maximowiczi and threw the beetles using their mandibles. Such counterattacks completely defended against attackers. The sounds that L. zenzeroides generated (pulse durations, 82–314 ms; dominant frequencies, 5.0–8.7 kHz; sound pressure level, 44.0–56.9 dB SPL) were produced by forcing air through the eighth pair of abdominal spiracles. Our results indicate that hornworm larvae are able to deter predacious invertebrates using multiple defences. INTRODUCTION Prey animals have evolved defensive behaviours against predators (Edmunds, 1974; Ruxton, Sherratt & Speed, 2004). During attack, some animals retaliate by biting, scratching, stinging, or discharging toxic chemicals that can damage the predators (Edmunds, 1974; Schmidt, 1990; Mukherjee & Heithaus, 2013). If the predators are injured by aggressive defences, they may give up their attack (cf. Sugiura & Yamazaki, 2014). Aggressive animal species that can injure predators are more likely to survive than are species that cannot. Thus, various types of aggressive defences have evolved in several animal taxa (Edmunds, 1974; Schmidt, 1990). Aggressive animal species may also warn their predators using their body colour, sounds, defensive devices and behaviours, thus forcing predators to avoid the prey (Schmidt, 1990). Caterpillars, including lepidopteran larvae, have multiple forms of defensive behaviour in response to predators (Greeney, Dyer & Smilanich, 2012). Larvae of the family Sphingidae (i.e. hornworms or hawkmoths) are relatively large and display aggressive defences against attackers (Walters et al., 2001; Brown, Boettner & Yack, 2007; Bura, Kawahara & Yack, 2016). For example, late instars of a hornworm species have a ‘defensive strike’ that includes rapid bending that accurately propels the head towards the abdominal site stimulated by attackers (Walters et al., 2001; Simon & Trimmer, 2009; van Griethuijsen, Banks & Trimmer, 2013). Multiple strikes (thrashing) can decrease the success of birds biting caterpillars (Walters et al., 2001). Striking behaviour is also accompanied by opening of the mandibles and occasional regurgitation (Walters et al., 2001); regurgitants can function as chemical defences against predators (Grant, 2006; Brown et al., 2007; Greeney et al., 2012). Furthermore, late instars of several hornworm species produce sounds in response to physical stimuli (Brown et al., 2007; Bura et al., 2011, 2012, 2016), which can deter vertebrate predators, such as birds (Brown et al., 2007; Bura et al., 2011; Dookie et al., 2017). Vertebrates, including birds, are important predators of large caterpillars in natural conditions (Stewart, 1975; Remmel, Davison & Tammaru, 2011). Therefore, previous studies have focused on vertebrate predators as important agents for providing selective pressures on the evolution of aggressive defences in caterpillars belonging to the superfamily Bombycoidea (including the families Sphingidae and Saturniidae; Brown et al., 2007; Bura et al., 2011, 2016). Arthropod predators can also pose strong predation pressures on hornworm larvae (Madden & Chamberlin, 1945; Lawson, 1959). Although hornworms frequently encounter invertebrate predators and parasitoids on host plants and on the ground (cf. Madden & Chamberlin, 1945; Bellotti, Arias & Guzman, 1992), it is unclear whether hornworms show aggressive defences (i.e. striking behaviour, regurgitation and/or sound production) against invertebrate predators. Could hornworm defences be effective against invertebrate attackers? Defensive strikes and regurgitation can function as behavioural and chemical defences, respectively, against attackers (Walters et al., 2001; Brown et al., 2007). Some invertebrate predators may respond to sounds and/or substrate-borne vibrations produced by hornworm larvae (Bura et al., 2016), although many invertebrate predators lack tympanal ears that are sensitive to sounds with broad frequencies (Yager, 1999; Yack, 2004). Determining the defences of hornworms in response to invertebrate attackers would improve our understanding of how defensive behaviours have evolved in caterpillars. To determine the function and mechanism of hornworm defences against invertebrate attackers, we investigated such behaviour in the hornworm Langia zenzeroides Moore (Lepidoptera: Sphingidae) in response to attacks by the caterpillar-feeding beetle Calosoma maximowiczi Morawitz (Coleoptera: Carabidae). Langia zenzeroides is the largest hornworm in Japan (body length of last instars: 100–130 mm; Yasuda, 2010; Kishida, 2011) and produces sounds in the larval, prepupal and adult stages (Inoue et al., 1982). In addition, the phylogenetic position of L. zenzeroides is unique in the plesiomorphic condition of the two subfamilies (i.e. Smerinthinae and Sphinginae; Kawahara et al., 2009). Therefore, L. zenzeroides is a model hornworm for investigating how aggressive defences have evolved in the family Sphingidae. The adults of the carabid genus Calosoma are well known as caterpillar hunters (Bruschi, 2013; Toussaint & Gillett, 2017), providing an appropriate predator for investigating the defensive behaviour of caterpillars (Sugiura & Yamazaki, 2014; Sugiura, 2016). Thus, we observed the responses of L. zenzeroides larvae to C. maximowiczi attacks in laboratory conditions. In addition, we characterized and clarified the mechanisms of sound production by the hornworm. Finally, we discuss the effectiveness of aggressive defences in hornworm caterpillars. MATERIAL AND METHODS Study species In this study, L. zenzeroides larvae were reared from eggs obtained from two female adults that were captured in Takarazuka, Hyogo Prefecture, central Japan (34°51′N, 134°18′E, 130 m above sea level) in early April 2015. Langia zenzeroides grows through five larval instars before pupating (Yasuda, 2010). The instar was determined based on the maximal width of the head capsule (first instar, 1.4–1.5 mm; second instar, 1.9–2.2 mm; third instar, 2.9–3.1 mm; fourth instar, 4.4–4.6 mm; fifth instar, 6.1–6.6 mm). Larvae were reared on leaves of Cerasus × yedoensis (Matsum.) A.V. Vassil. and Cerasus speciosa (Koidz.) H. Ohba (Rosaceae) in laboratory conditions (25 °C, 16 h–8 h light–dark cycle). Fifth instars of L. zenzeroides produced sounds in response to physical stimuli (see Supporting information, Movie S1). In laboratory experiments, we used larvae that were randomly chosen from among 37 fifth instars of L. zenzeroides (body length, mean ± SEM, 100.4 ± 2.1 mm). Several larvae were used in different experiments. Before experimentation, the body length of each hornworm was measured to the nearest 0.1 mm using slide callipers while it was resting on a twig. Calosoma maximowiczi adults attack multiple caterpillar species on the ground and in vegetation (Sugiura & Yamazaki, 2014; Sugiura, 2016). As found in some predatory animals (e.g. Sugiura et al., 2011; Ohba & Tatsuta, 2016), C. maximowiczi adults were able to attack prey larger than themselves (Sugiura, 2016). For the following experiments, all of the adults of C. maximowiczi were collected from a secondary forest in Kobe, Hyogo (34°42′N, 134°11′E, 60–170 m above sea level), in early May 2015. Although we have not observed C. maximowiczi attacking hornworms in field conditions, the habitat and active season overlap between C. maximowiczi adults and L. zenzeroides larvae. Therefore, L. zenzeroides larvae might encounter C. maximowiczi on host plants or on the ground. Twenty-five C. maximowiczi adults were used in this study, and the body length of each adult was measured to the nearest 0.1 mm using slide callipers. Behavioural experiment To elucidate defensive behaviour against invertebrate attackers, we investigated the response of L. zenzeroides larvae to bites and other physical contact by the invertebrate attacker C. maximowiczi. The experiment was performed during the day in May 2015 in a well-lit laboratory (25 ± 1 °C). Given that C. maximowiczi adults and L. zenzeroides larvae naturally forage on twigs and trunks, a carabid adult and a hornworm were placed on bamboo (width, 7 mm; height, 15 mm; Fig. 1; cf. Sugiura 2016). The bamboo was looped (length, 700 mm; diameter, 200 mm; Fig. 1) such that hornworms could encounter carabids in all trials. The looped bamboo was also surrounded by a plastic circular cylinder (diameter, 220 mm; height, 120 mm; Fig. 1). We used 25 L. zenzeroides larvae (body length, mean ± SEM, 105.4 ± 1.9 mm) and 25 C. maximowiczi adults in the experiment. Figure 1. View largeDownload slide The arena used in our experiments. (A) A Langia zenzeroides larva and a Calosoma maximowiczi adult placed on bamboo material (Sugiura, 2016). (B) Overhead view of the arena (looped bamboo material) surrounded by a plastic circular cylinder. Scale bars: 15 mm. Figure 1. View largeDownload slide The arena used in our experiments. (A) A Langia zenzeroides larva and a Calosoma maximowiczi adult placed on bamboo material (Sugiura, 2016). (B) Overhead view of the arena (looped bamboo material) surrounded by a plastic circular cylinder. Scale bars: 15 mm. Immediately before the experiment, the activity of C. maximowiczi was assessed using the prey caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) (sixth instar, 22.0–35.0 mm in length). We used forceps to move prey caterpillars in front of C. maximowiczi. When C. maximowiczi attacked (bit) prey caterpillars, we considered them to be active. Eleven C. maximowiczi (four males and seven females; body length, mean ± SEM, 25.7 ± 0.5 mm) attacked prey caterpillars (hereafter, ‘active beetles’), and 14 adults did not (hereafter, ‘inactive beetles’; six males and eight females; body length, mean ± SEM, 26.7 ± 0.4 mm). The sex ratios and body lengths were not significantly different between active and inactive beetles (sex ratio, Fisher’s exact test, P = 1.0; body length, t-test, t = −1.4443, P = 0.16). All active beetles bit prey with their mandibles. Although inactive adults did not bite prey, their body parts frequently touched the prey bodies. Thus, we used active and inactive C. maximowiczi adults to investigate the defensive responses of L. zenzeroides larvae to bites and other physical touches (without bites) by C. maximowiczi, respectively. First, we placed a hornworm on bamboo. When the hornworm rested on the bamboo, a carabid adult was placed on the far end of the bamboo (Fig. 1). During a 10 min period, recordings were obtained for whether a carabid beetle used mandibles to attack L. zenzeroides, how many times the carabid contacted the larva, how the larva responded to the contacts (i.e. strike, sound production and/or regurgitation) and how the carabid responded to L. zenzeroides defences (i.e. retreat or continue to attack). We also recorded the body part (head, thorax, abdomen A1–3, A4–7 or A8–10) touched or bitten by the carabid and the responses in each L. zenzeroides larva. After an L. zenzeroides larva successfully repelled a beetle, we continued to watch for further attacks by C. maximowiczi within the 10 min period (cf. Sugiura, 2016). Defensive behaviour was recorded using the movie function of a digital camera (iPhone 6 plus; Apple) at 240 frames s−1. Sound and video recordings The sounds produced by randomly chosen larvae (body length, mean ± SEM, 89.0 ± 4.8 mm; N = 9) were recorded. Two of nine larvae were also used in the behavioural experiment. The recordings were made before the behavioural experiment. Each larva was placed on a cut twig, which was perpendicular to the ground, so that the larva hung its head down from the posterior prolegs. Larvae were stimulated from both sides of the abdomen (A8–A10) using forceps. During these trials, the larvae produced sounds, which were recorded using a 1/4-inch condenser microphone (type 4939; Brüel & Kjær, Nærum, Denmark) in a soundproof room. Sound signals were amplified (type 2670 and 2690, with a 0.02–100 kHz bandpass filter; Brüel and Kjær) and digitized using an analog converter with a 0.7 Hz high-pass filter (PULSE type 3560-B; Brüel & Kjær) at a sampling rate of 65.5 kHz (24 bits). The digitized sounds were analysed using PULSE Labshop software (version 15.1.0; Brüel & Kjær). The microphone was held at a distance of 20 mm from the dorsal surface of the larval abdomen, and power spectra were computed in the PULSE Reflex software (version 18.1.1; Brüel & Kjær) using a Hanning window (fast Fourier transform, line, 400; frequency resolution, 128 Hz; frequency span, 51.2 kHz). The spectral characteristics were analysed with a high-pass filter at 500 Hz. The average and individual spectra of sounds recorded from nine larvae were calculated after the conversion of logarithmic decibels into linear micropascals. Sound levels were determined as the decibel peak equivalent sound pressure level (dB peSPL; 0 dB = 20 μPa rms), referring to signals from a sound level calibrator (type 4230, 94 dB SPL at 1 kHz; Brüel & Kjær). A pulse is defined as a group of uninterrupted waves or elements (Bura et al., 2011). Temporal characters of pulse durations and dominant frequencies were measured from the recordings of nine larvae. Similar methods have been used in previous studies (cf. Takanashi et al., 2010; Bura et al., 2011; Tsubaki et al., 2014). The strike behaviour of randomly chosen larvae (body length, mean ± SEM, 87.2 ± 5.1 mm; N = 7) was filmed using a high-speed camera (FASTCAM Mini UX50; Photron, Japan) with a Zoom-Nikkor lens (35–70 mm, f/3.3–4.5; Nikon, Japan) at 500 frames s−1. We measured the time required from the start of movement to striking the head against the forceps. When multiple strikes occurred, we measured the duration of the first strike. Sounds were recorded at the same time using the type 4939 microphone placed 20 mm away from the dorsal abdomen. Signals from the microphone were transmitted as described above, and the recorded videos were combined with the audio (FASTCAM Analysis, version 1.2.1.1; Photron, Japan) and analysed temporally. Sound production mechanism To clarify the mechanisms leading to sound production in L. zenzeroides larvae, we tested ‘clicking’ by the mandibles (Brown et al., 2007; Bura et al., 2012), ‘whistling’ through the abdominal spiracles (Bura et al., 2011) and ‘vocalizing’ through the oral cavity (Bura et al., 2016). First, larval mandibles were observed using the digital camera (iPhone 6 plus) during sound production, and the movement of seven larvae (body length, mean ± SEM, 89.9 ± 5.2 mm) was played back using QuickTime Player version 10.4 (Apple, Inc.). Second, ten larvae (body length, mean ± SEM, 97.9 ± 3.0 mm) were individually submerged in a plastic container (diameter, 155 mm; height, 60 mm) filled with water (300 mL) and pinched with forceps. Behaviour was filmed using a video camera (Handycam, HDR-CX630; Sony, Japan), and the QuickTime Player was used to check whether air bubbles emerged from abdominal spiracles, the mouth or other body locations. Statistical analysis Fisher’s exact tests were performed to compare the frequency of strike behaviour, sound production and regurgitation between different responses (i.e. responses to active or inactive beetles). A generalized linear model (GLM) with Poisson error distribution and log link was used to clarify the effects of attacker types (i.e. active or inactive beetles) on the number of contacts with L. zenzeroides larvae. The number of physical contacts between beetle attackers and L. zenzeroides larvae was used as a response variable, whereas active and inactive beetles were treated as fixed factors. A quasi-Poisson error distribution was used when the residual deviance was larger than the residual degrees of freedom (i.e. overdispersion; Crawley, 2005). All the analyses were performed using R version 3.2.2 (R Development Core Team, 2015). RESULTS Defensive behaviour in response to carabid beetles The number of physical contacts with L. zenzeroides larvae ranged from 12 to 38 and from seven to 48 in active and inactive beetles, respectively, which was not significantly different (GLM, taking into account overdispersion, t = −1.143, P = 0.25). Heads, thoraxes and/or abdomens of L. zenzeroides larvae were touched or bitten by the beetles (see Supporting information, Table S1). All the active beetles attacked L. zenzeroides larvae that were 3.4–4.6 times longer, and all active beetles used their mandibles to bite the heads or abdomens of L. zenzeroides larvae at least once (Fig. 2A). All L. zenzeroides larvae (N = 11) whose heads, thoraxes or abdomens were stimulated by carabids exhibited striking responses to active beetles by bending their heads towards the location that was bitten or contacted (Fig. 2B, Table 1; see Supporting information, Table S2). Langia zenzeroides larvae also exhibited multiple strikes (thrashing) while being repeatedly attacked by carabid beetles (see Supporting information, Movie S2). All the larvae (N = 11) exhibiting strike behaviour simultaneously produced sounds that were audible to the human ear (Table 1); sound production was confirmed when heads, thoraxes and/or abdomens of L. zenzeroides larvae were attacked by the beetles (see Supporting information, Table S3). Regurgitation was also accompanied by strike behaviour and sound production in 54.5% (N = 6/11) of the larvae that were bitten by beetles (Table 1). Leaf fragments were included in the regurgitant. Two larvae (18.2%) caught the legs of beetles using their mandibles and threw them. Additionally, one of the two larvae removed the distal portion of the beetle’s right hindleg (tibia and tarsus) by biting (Fig. 3; see Supporting information, Movie S2). No beetles killed L. zenzeroides larvae, and two active beetles retreated in response to L. zenzeroides strikes and bites. Therefore, such counterattacks could effectively defend L. zenzeroides against carabid attacks. Table 1. Defensive behaviours of Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi   Percentage of L. zenzeroides larvae (N)*  Behavioural responses  Bites and/or contacts by active beetle  Physical contacts by inactive beetle  Strike, sound and regurgitation  54.5 (6)  7.1 (1)  Strike and sound  45.5 (5)  42.9 (6)  Strike and regurgitation  0.0 (0)  0.0 (0)  Strike (only)  0.0 (0)  35.7 (5)  Sound and regurgitation  0.0 (0)  0.0 (0)  Sound (only)  0.0 (0)  0.0 (0)  Regurgitation (only)  0.0 (0)  0.0 (0)  None  0.0 (0)  14.3 (2)  Total  100.0 (11)  100.0 (14)    Percentage of L. zenzeroides larvae (N)*  Behavioural responses  Bites and/or contacts by active beetle  Physical contacts by inactive beetle  Strike, sound and regurgitation  54.5 (6)  7.1 (1)  Strike and sound  45.5 (5)  42.9 (6)  Strike and regurgitation  0.0 (0)  0.0 (0)  Strike (only)  0.0 (0)  35.7 (5)  Sound and regurgitation  0.0 (0)  0.0 (0)  Sound (only)  0.0 (0)  0.0 (0)  Regurgitation (only)  0.0 (0)  0.0 (0)  None  0.0 (0)  14.3 (2)  Total  100.0 (11)  100.0 (14)  *Values in parentheses indicate the numbers of L. zenzeroides larvae. View Large Figure 2. View largeDownload slide The hornworm Langia zenzeroides and its potential attacker Calosoma maximowiczi. (A) An adult C. maximowiczi biting the head of an L. zenzeroides larva. (B) An L. zenzeroides larva striking an adult C. maximowiczi. (C) A fifth instar of L. zenzeroides. One and eight pairs of spiracles are paced on the thorax (T1) and abdomen (A1–A8), respectively. The arrow indicates the spiracle on the eighth abdominal segment (A8). Scale bars: 10 mm. Figure 2. View largeDownload slide The hornworm Langia zenzeroides and its potential attacker Calosoma maximowiczi. (A) An adult C. maximowiczi biting the head of an L. zenzeroides larva. (B) An L. zenzeroides larva striking an adult C. maximowiczi. (C) A fifth instar of L. zenzeroides. One and eight pairs of spiracles are paced on the thorax (T1) and abdomen (A1–A8), respectively. The arrow indicates the spiracle on the eighth abdominal segment (A8). Scale bars: 10 mm. Figure 3. View large Download slide Sequential images of Langia zenzeroides behaviour in response to Calosoma maximowiczi (see Supporting information, Movie S2). The strike (150 ms), bite (450 ms), throw (600–750 ms) and regurgitation times (600–800 ms) were observed. The arrows indicate the beetle leg removed by the larva. Scale bar: 15 mm. Figure 3. View large Download slide Sequential images of Langia zenzeroides behaviour in response to Calosoma maximowiczi (see Supporting information, Movie S2). The strike (150 ms), bite (450 ms), throw (600–750 ms) and regurgitation times (600–800 ms) were observed. The arrows indicate the beetle leg removed by the larva. Scale bar: 15 mm. All inactive beetles touched L. zenzeroides larvae but not to bite them. Physical contacts by inactive beetles stimulated 85.7% of the larvae (N = 12/14) to evoke striking responses (Table 1). Sound production accompanied striking behaviour in 58.3% (N = 7/12) of the larvae (Table 1), and no responses were observed in 14.3% of the larvae touched by inactive beetles (Table 1). Only one larva regurgitated, when its posterior abdomen was merely touched by an inactive beetle (Table 1; see Supporting information, Table S4). The frequency of strike behaviour in larvae touched by inactive beetles was not significantly different from that of larvae bitten by active beetles (Fisher’s exact test; sound production, P = 0.49). However, larvae bitten by active beetles produced sound and regurgitated more frequently than larvae contacted by inactive beetles (Fisher’s exact test; sound production, P = 0.0078; regurgitation, P = 0.0213). Strike behaviour, sound characteristics and mechanism of sound production Langia zenzeroides larvae performed a striking response to abdominal pinching by forceps, in which they rapidly bent their heads and thoraxes towards the pinched abdomen (see Supporting information, Movie S1). The duration of the behaviour (from start of bending to striking) ranged from 142 to 230 ms (mean ± SEM, 180.6 ± 13.0 ms; N = 7; see Supporting information, Movie S3). The larvae produced a single sound pulse in response to artificial stimuli (see Supporting information, Movie S3, Audio S1). The pulse duration ranged from 82 to 314 ms (mean ± SEM, 166.7 ± 21.1 ms; N = 9; Fig. 4A), and spectral analyses revealed that the dominant frequency ranged from 5.0 to 8.7 kHz (mean ± SEM, 5.9 ± 0.4 kHz; N = 9; Fig. 4B), and the maximal sound pressure ranged from 44.0 to 56.9 dB SPL (mean, 49.8 dB SPL, N = 9; Fig. 4B). Larval mandibles (N = 7) remained open during sound production, suggesting that L. zenzeroides does not produce sound with their mandibles. When L. zenzeroides larvae were submerged in water, 30% (N = 3/10) produced air bubbles from a pair of spiracles on the eighth abdominal segment with sounds that were audible to the human ear (Fig. 2C; see Supporting information, Movie S4), indicating that L. zenzeroides larvae ‘whistle’ through a pair of eighth abdominal spiracles. The lack of sound production in some larvae was probably the result of being submerged in water. Figure 4. View large Download slide Characteristics of the sounds produced by Langia zenzeroides larvae. (A) An oscillogram of the sound pulse produced by a larva (see Supporting information, Audio S1). (B) Power spectra of larval pulses. The black and red lines are derived from the pulse of the larva (shown in the oscillogram) and the pulses of nine larvae (i.e. means of N = 9), respectively. Figure 4. View large Download slide Characteristics of the sounds produced by Langia zenzeroides larvae. (A) An oscillogram of the sound pulse produced by a larva (see Supporting information, Audio S1). (B) Power spectra of larval pulses. The black and red lines are derived from the pulse of the larva (shown in the oscillogram) and the pulses of nine larvae (i.e. means of N = 9), respectively. DISCUSSION Aggressive defences that can damage or kill attackers have been reported in some animal species (Edmunds, 1974; Schmidt, 1990; Mukherjee & Heithaus, 2013). Lepidopteran larvae show various types of aggressive defences, including defensive strikes, sound production and regurgitation (Walters et al., 2001; Brown et al., 2007; Greeney et al., 2012; Bura et al., 2016). However, only a few studies have focused on which stimuli cause aggressive defences and how the aggressive defences might be effective (Walters et al., 2001; Dookie et al., 2017). In the present study, we showed that C. maximowiczi attacks caused L. zenzeroides larvae to exhibit striking behaviour and/or sound production (Table 1). Beetle bites also frequently induced regurgitation (Table 1). Therefore, such aggressive defences could effectively protect L. zenzeroides larvae from beetle attack, increasing larval survivorship. Counterattacks The number of physical contacts with L. zenzeroides larvae did not differ between active and inactive beetles. In addition, the frequency of strike behaviour in larvae touched by inactive beetles was not different from that of larvae bitten by active beetles (Table 1). These results suggest that L. zenzeroides larvae exhibit strike behaviour in response to almost all physical contact with beetles (including biting). Although many hornworm species exhibit strike behaviour (Edmunds, 1974; Wagner, 2005), detailed assessments of such behaviours have been performed only in Manduca sexta (L.) (Walters et al., 2001; Simon & Trimmer, 2009; van Griethuijsen et al., 2013). Walters et al. (2001) showed that thrashing (repeated strikes) of M. sexta larvae decreased the success of avian predators. Although the strike behaviour of M. sexta larvae is thought to dislodge smaller predators (Walters et al., 2001), hornworm defences against predacious invertebrates have remained unexplored. Langia zenzeroides larvae counterattacked C. maximowiczi by striking (Fig. 3; see Supporting information, Movie S2), suggesting that hornworm strikes function as a behavioural defence against predacious invertebrates. In addition, L. zenzeroides larvae threw C. maximowiczi when legs were lodged in their mandibles, and it is surprising that the soft-bodied caterpillar injured the hard-bodied beetle (Fig. 3; see Supporting information, Movie S2). Although some hornworm species nip at their attackers (Wagner, 2005), such an aggressive defence has rarely been reported. Langia zenzeroides larvae bitten by active beetles produced sound and regurgitated more frequently than larvae contacted by inactive beetles, although larvae contacted by inactive beetles exhibited strike behaviour as frequently as those bitten by active beetles (Table 1). Therefore, beetle contact stimuli caused L. zenzeroides larvae to exhibit a single defence (strike behaviour), whereas intense stimuli (i.e. beetle bites) induced L. zenzeroides larvae to exhibit multiple defences (strikes, sound production and regurgitation). Leaf fragments included in the larval regurgitants suggest that L. zenzeroides larvae regurgitated their gut contents; such regurgitants are considered chemical defences against predators (Grant, 2006; Brown et al., 2007; Greeney et al., 2012). Collectively, the multiple defences of L. zenzeroides larvae routinely repelled carabid attackers (Fig. 3; see Supporting information, Movie S2), although the costs of multiple defences are higher than those of a single defence (strike behaviour). Given that only one insect species (i.e. C. maximowiczi) was used as a model predator in this study, the defensive responses of L. zenzeroides larvae against other types of predators remain unclear. Various enemies might attack L. zenzeroides larvae in field conditions, although no enemies have been recorded. Therefore, further experiments are needed to clarify the effectiveness of L. zenzeroides defences against invertebrate predators and parasitoids. Sound production and its mechanism Sound production has been reported in all three subfamilies of Sphingidae (i.e. Macroglossinae, Sphinginae and Smerinthinae; Bura et al., 2016). In two subfamilies, Macroglossinae and Sphinginae, clicking of the mandibles produces sound. Vocalizing through the oral cavity occurs only in Macroglossinae, and whistling through the abdominal spiracles occurs in Smerinthinae (Bura et al., 2016). Our data revealed that L. zenzeroides larvae produce sound by forcing air through a pair of eighth abdominal spiracles (Fig. 2C; see Supporting information, Movie S4). A similar mechanism has been reported in the smerinthine caterpillar Amorpha juglandis (Smith), although the dominant frequencies and sound pressure levels of L. zenzeroides larvae were marginally lower than those of A. juglandis larvae (Bura et al., 2011). Kawahara et al. (2009) determined that the placement of L. zenzeroides within the phylogeny is unique in that it illustrates the plesiomorphic condition of the Smerinthinae and Sphinginae. Given that sound production is known in only two smerinthines (Bura et al., 2016), and because Langia and Amorpha are not closely related, it appears that these similar mechanisms might have arisen independently. The sounds produced by hornworm larvae can frighten vertebrate predators (startle displays; Brown et al., 2007; Bura et al., 2016) or warn them (acoustic aposematism; Bura et al., 2011, 2016; Dookie et al., 2017); however, the defensive function against invertebrates remains unclear. Some coleopteran insects, including cicindelines and scarabaeids, have tympanal ears that are sensitive to sounds with broad frequencies (Yager, 1999; Yack, 2004). Whether the carabid beetle C. maximowiczi has tympanal ears to detect sounds made by L. zenzeroides remains unclear. However, C. maximowiczi might be able to detect substrate-borne vibrations produced by L. zenzeroides (cf. Autrum & Schneider, 1948). In addition, many L. zenzeroides larvae that produced sounds simultaneously exhibited strike behaviour and/or regurgitation (Table 1). Given that defensive strikes and regurgitants can damage predacious invertebrates, L. zenzeroides sounds might function as a warning against invertebrate attackers. However, our results did not fully clarify the defensive functions of L. zenzeroides sounds against invertebrate attackers, and the responses of vertebrate attackers to L. zenzeroides sounds remain unclear. Additional experiments are required to determine the defensive functions of L. zenzeroides sounds. CONCLUSION Although the strike behaviours of hornworm caterpillars startle vertebrate predators (Walters et al., 2001), the present study indicated that L. zenzeroides strikes deter predacious beetles. This is the first demonstration of the defensive functions of hornworm strikes against predacious invertebrates. Multiple defences, including strikes, throwing, regurgitation and sound production, could effectively discourage invertebrate attackers. ACKNOWLEDGEMENTS We thank T. Ohshio and I. Hanatani for providing L. zenzeroides adults and assistance with the rearing of caterpillars, respectively. We also thank the Editor and several reviewers for their valuable comments on our manuscript. This research was partly supported by Grants-in-Aid for Scientific Research (26450065 and 17K08158). Our experiments were conducted in accordance with the Kobe University Animal Experimentation Regulations (Kobe University’s Animal Care and Use Committee, H27). The experiments also complied with the current laws of Japan. The authors declare there are no competing financial interests. S.S. and T.T. conceived and designed experiments. S.S. collected and reared insects and performed behavioural experiments. S.S. and T.T. carried out sound recording and analyses and analysed the data. S.S. wrote the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Numbers of contacts between Langia zenzeroides larvae and Calosoma maximowiczi adults. Table S2. Percentage of strike behaviour in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Table S3. Percentage of sound production in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Table S4. Percentage of regurgitation in Langia zenzeroides larvae in response to the carabid beetle Calosoma maximowiczi. Movie S1. Strike behaviour of Langia zenzeroides larvae in response to artificial stimuli (i.e. pinched by forceps). Movie S2. Defensive behaviour of Langia zenzeroides larvae in response to physical contacts or attacks by Calosoma maximowiczi. Movie S3. Slow-motion video of strike and sound production by Langia zenzeroides in response to an artificial stimulus. The vertical axis indicates the timing of the left video of the strike, in the right figure, without a high-pass sound filter applied to the oscillogram. Movie S4. Mechanism of sound production in Langia zenzeroides. 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