Introduction
Tonic immobility, also known as death-feigning or thanatosis, is a behavioural trait in which prey adopt a motionless state in a characteristic posture elicited by external stimuli (Humphreys and Ruxton, Reference Humphreys and Ruxton2018; Sakai, Reference Sakai2021). Several hypotheses have been proposed to explain the adaptive value of this behaviour (Miyatake et al., Reference Miyatake, Katayama, Takeda, Nakashima, Sugita and Mizumoto2004, Reference Miyatake, Nakayama, Nishi and Nakajima2009; Ruxton, Reference Ruxton2006). For example, it can increase survival after detection by visual predators that are better able to detect moving prey, as these predators are less likely to attack immobile prey (Miyatake et al., Reference Miyatake, Katayama, Takeda, Nakashima, Sugita and Mizumoto2004, Reference Miyatake, Nakayama, Nishi and Nakajima2009). Some of the adaptive value of this behaviour is specific to certain predator types; for example, grasshoppers adopt a rigid posture during tonic immobility to avoid being swallowed whole by gape-limited predators (Honma et al., Reference Honma, Oku and Nishida2006).
Although tonic immobility has been documented in many terrestrial taxa, including both vertebrates and invertebrates (Humphreys and Ruxton, Reference Humphreys and Ruxton2018), there are relatively few studies that have quantified tonic immobility in marine invertebrates. Behaviour resembling tonic immobility is frequently documented in decapod crustaceans in both freshwater and marine environments (Mackay, Reference Mackay1929; Powell and Gunter, Reference Powell and Gunter1968; O'Brien and Dunlap, Reference O'Brien and Dunlap1975; Field, Reference Field1990; Bergey and Weis, Reference Bergey and Weis2006; Scarton et al., Reference Scarton, Zimmermann, Machado, Aued, Manfio and Santos2009; Coutinho et al., Reference Coutinho, Ayres-Peres, Araujo, Jara and Santos2013). Tonic immobility in decapod crustaceans has been documented primarily in species targeted by bird and fish predators, and its occurrence as a response against nemertean predators has also been observed (Christy et al., Reference Christy, Goshima, Backwell and Kreuter1997; Hazlett and McLay, Reference Hazlett and McLay2000; Bergey and Weis, Reference Bergey and Weis2006).
The frequency and duration of tonic immobility can be influenced by individual characteristics such as physiological context (Miyatake, Reference Miyatake2001a; Krams et al., Reference Krams, Kivleniece, Kuusik, Krama, Freeberg, Mänd, Vrublevska, Rantala and Mänd2013), sex (Miyatake, Reference Miyatake2001a, Reference Miyatake2001b; Kuriwada et al., Reference Kuriwada, Kumano, Shiromoto and Haraguchi2009), and body size (Hozumi and Miyatake, Reference Hozumi and Miyatake2005; Farkas, Reference Farkas2016). However, these influences are not consistent across taxa. For example, whereas increased body size is associated with prolonged tonic immobility in a weevil (Hozumi and Miyatake, Reference Hozumi and Miyatake2005), small body size is associated with more frequent tonic immobility in a stick insect (Farkas, Reference Farkas2016). Similarly, females exhibit prolonged tonic immobility in comparison to males in a seed beetle (Miyatake et al., Reference Miyatake, Okada and Harano2008), but a shorter period of immobility in a leaf beetle (Mueller and Mueller, Reference Mueller and Mueller2017). In the marine system, the duration of tonic immobility by the rock crab Heterozius rotundifrons varies with chemical and visual cues (Field, Reference Field1990; Hazlett and McLay, Reference Hazlett and McLay2000, Reference Hazlett and McLay2005), but is not significantly affected by body size or sex (Bach and Hazlett, Reference Bach and Hazlett2010).
Marine isopods are targeted by various predators including flatworms, crabs, birds, and fish in intertidal and subtidal zones (Wallerstein and Brusca, Reference Wallerstein and Brusca1982; Leidenberger et al., Reference Leidenberger, Harding and Jonsson2012). They possess various defensive traits, including microhabitat choice (Merilaita and Jormalainen, Reference Merilaita and Jormalainen1997; Vesakoski et al., Reference Vesakoski, Merilaita and Jormalainen2008), chemical defence (Lindquist et al., Reference Lindquist, Barber and Weisz2005), and cryptic colouration (Merilaita, Reference Merilaita1998; Hultgren and Mittelstaedt, Reference Hultgren and Mittelstaedt2015). Sexual differences in predator avoidance response are well-studied in Idotea baltica (Jormalainen and Tuomi, Reference Jormalainen and Tuomi1989; Merilaita and Jormalainen, Reference Merilaita and Jormalainen1997; Vesakoski et al., Reference Vesakoski, Merilaita and Jormalainen2008). Idotea baltica females reduce their activity during reproduction in the face of high predation risk, whereas males retain high activity despite the risk because they prioritize mate search over predator avoidance (Jormalainen & Tuomi Reference Jormalainen and Tuomi1989; Vesakoski et al., Reference Vesakoski, Merilaita and Jormalainen2008). This higher risk aversion in females relative to males is thought to reflect differences in their strategies for maximizing reproductive success (Vesakoski et al., Reference Vesakoski, Merilaita and Jormalainen2008).
The marine isopod Cleantiella isopus is a common intertidal benthic species found under rocks and on algae on the intertidal rocky shore. Cleantiella isopus has a lifespan of 13–15 months from birth (at a body size of about 5 mm) to death. In our study area, they usually have three breeding periods: from late February to mid-April and from late May to early June, with some individuals breeding again in July (Takahashi and Goshima, Reference Takahashi and Goshima2012). This species exhibits tonic immobility with an arch-like posture and motionless state in response to external stimuli such as attacks by predatory fish (Figure 1). To our knowledge, no studies have previously documented tonic immobility in aquatic isopods, although some reports exist for terrestrial isopods (Quadros et al., Reference Quadros, Bugs and Araujo2012; Tuf et al., Reference Tuf, Drábková and Šipoš2015; Cazzolla Gatti et al., Reference Cazzolla Gatti, Messina, Tiralongo, Ursino and Lombardo2020). In this paper, we describe the tonic immobility of C. isopus and examine the influence of body size, sex, and colour morph on the frequency and duration of tonic immobility.
Figure 1. Examples of Cleantiella isopus posture (A) during tonic immobility and (B) after tonic immobility. The ruler in the background is in units of centimetres.
Materials and methods
Study animals
The population of C. isopus used in this study consists of five distinct colour morphs which can be classified as brown, brown with a white line, brown with a white spot, light brown, and green (Takahashi and Goshima, Reference Takahashi and Goshima2012). Three of the brown-based morphs appear to be well concealed in brown algae such as Neorhodomela aculeata, whereas the light brown and green morphs are well concealed on sandy substratum. Patterns of microhabitat utilization are similar among the colour morphs and between the two sexes, since these categories often coexist within the same shelter under stones during low tide (Takahashi and Goshima, Reference Takahashi and Goshima2012).
Individuals were collected from an intertidal rocky shore in Kattoshi (41°44′N, 140°36′E) on the western side of Hakodate Bay, southern Hokkaido, Japan, during low tides in April and May 2022, the breeding season of C. isopus (Takahashi and Goshima, Reference Takahashi and Goshima2012). In total, we collected 230 individuals, including 159 females and 71 males, with body length ranging from 14.1 mm to 36.7 mm. They were brought back to the laboratory and individually placed in a small plastic container (130 × 95 × 65 mm, L × W × H) filled with natural seawater (approximately 30 mm in depth) at ambient water temperature (approximately 13 °C). The following observations were conducted after an acclimation period of one hour. After the experiment, all individuals were released back into the field site from which they were collected.
Observation of tonic immobility
The procedure for the behavioural trials was based on previous studies of terrestrial isopods (Quadros et al., Reference Quadros, Bugs and Araujo2012). Tonic immobility was induced by poking each isopod five times with a stick to simulate an attack by a predatory fish. This stimulus was specifically intended to imitate immobilization by intertidal fish species (e.g. Chaenogobius annularis, Pholis crassispina), which are common predators in our study area. Preliminary observations indicated that the posture shown during tonic immobility prevents the isopods from being swallowed whole by fish, leading to repeated attacks from fish attempting to swallow them (Supplementary Video S1). If the isopods in our behavioural trials responded to the five consecutive pokes by adopting the posture associated with tonic immobility (Figure 1), the duration of immobility was recorded using a stopwatch. The duration was defined as the time elapsed between the end of the stimulation and the time when the individual moved slightly, which typically began with a movement of the second antennae. If the individual did not exhibit tonic immobility, the result was recorded as ‘no response’. Because the duration of tonic immobility in preliminary observations was mostly between 60 and 120 s, we observed the behaviour of each isopod for 300 s. Each individual was tested three times, with each test separated by approximately 30 min.
Body length, sex, and colour morph were recorded after the third behavioural trial. Body length was measured as the distance from the anterior border of the cephalon to the posterior border of the telson (Takahashi and Goshima, Reference Takahashi and Goshima2012; Miura and Goshima, Reference Miura and Goshima2016). The sex of each individual was identified by using a stereo microscope on the basis of the presence (male) or absence (female) of genital papillae on the underside of the pleon. The experiments were conducted in the early part of the breeding season, and all females in our study had brood pouches containing embryos in an early stage of development (Takahashi and Goshima, Reference Takahashi and Goshima2012).
Statistical analysis
We constructed a generalized linear mixed model (GLMM) with gamma error distribution and log link function for the duration of tonic immobility. Individual ID was included as a random effect in the model to account for nonindependence among the three trials for each individual. We included body length, sex (male or female), and colour morph (brown, brown with a white line, brown with a white spot, light brown, or green) as fixed effects. We included all possible interaction terms in the full model, but since the interaction terms were not significant, we did not include them in the final model. The significance of fixed effects was tested by Wald χ 2 tests. As males and females differed in body length, all data were segregated by sex (males, n = 71; females, n = 159). All statistical analyses were performed in R (version 4.2.1; R Core Team, 2022). Models were fitted using the glmmTMB function from the package glmmTMB (version 1.1.5; Brooks et al., Reference Brooks, Kristensen, van Benthem, Magnusson, Berg, Nielsen, Skaug, Maechler and Bolker2017). Fixed effects were tested using the Anova function from the package car (version 3.1.1; Fox and Weisberg, Reference Fox and Weisberg2019).
Results
All individuals (n = 230) exhibited tonic immobility in all trials, and the duration of tonic immobility was greater than 300 s in 19 out of the 690 total trials. We observed two distinct postures during tonic immobility: one in which the second antennae are extended (Figure 2A) and another in which the second antennae are folded (Figure 2B).
Figure 2. Two distinct postures of Cleantiella isopus were observed during tonic immobility: with the second antennae (A) extended or (B) folded. The scale bar is in units of centimetres.
There was a significant effect of body size on the duration of tonic immobility (df = 1, χ 2 = 37.305, P < 0.001; Table 1). Although a negative relationship was found between the duration of tonic immobility and body size (Estimate = –0.047 ± 0.008 SE; Figure 3), it depended on sex. The negative relationship was clear in males but not in females (Table 1; Figure 3). There was no significant effect of colour morph on the duration of tonic immobility (df = 4, χ 2 = 7.955, P = 0.116; Table 1).
Table 1. GLMM analysis of the duration of tonic immobility in Cleantiella isopus using gamma error distribution and log link function
Significant effects (at P ⩽ 0.05) are shown in bold.
Figure 3. Relationships between body size of Cleantiella isopus and the duration of tonic immobility for males (A) and females (B).
Discussion
All individuals exhibited tonic immobility, which is particularly impressive given the large number of individuals examined in this study. The high frequency of this behaviour implies that tonic immobility is a major response against predatory attack in C. isopus regardless of body size, sex, or colour morph. The arch-like posture adopted during tonic immobility may be an adaptation to predators that swallow their prey whole (Moore and Williams, Reference Moore and Williams1990; Honma et al., Reference Honma, Oku and Nishida2006). The high frequency of tonic immobility observed in this study could indicate a high lethality of predator attacks when isopods fail to assume the correct posture. Based on preliminary observations, we used five pokes with a stick as a stimulus to induce tonic immobility, but this stimulus may have been unnatural. Excessively strong stimulation could have caused the high frequency of tonic immobility observed in our study, but we believe this is unlikely because no individuals appeared to have been injured by the stimulation during the trials. Instead, we suggest that the uniformity of our stimulation method explains the uniformity of the observed response.
We found a negative relationship between overall body size and the duration of tonic immobility. This pattern was particularly evident in males. Male body size is quite variable (Takahashi and Goshima, Reference Takahashi and Goshima2012), and this variation might mean that there are large differences in predation risk among male C. isopus. In contrast, the range of female body size was small, indicating that factors other than body size are likely to be responsible for the observed variation in the duration of tonic immobility. We hypothesize that the negative relationship between the duration of tonic immobility and body size may reflect a decrease in predation risk with increasing body size. If this were the case, smaller individuals could derive a greater benefit from remaining immobile for longer periods. Smaller individuals may also be more elusive to a visual predator since they can become concealed among fragments of seaweed during periods of tonic immobility. A similar mechanism has been hypothesized for a stick insect (Farkas, Reference Farkas2016), where smaller individuals that exhibit tonic immobility are thought to have a greater chance of avoiding detection by a predator after falling to the ground. It is also possible that small and large C. isopus are preyed upon by predators of differing size and/or type. Several types of predators, including flatworms, crabs, and fish, are present in our study area (K. Igarashi, personal observation). Based on the hypotheses outlined above, we believe that tonic immobility is a specific behavioural defence against predation by fish that rely on visual cues for prey detection. In addition to being visual predators, most fish are gape limited, meaning they swallow their prey whole. The role of tonic immobility in isopods appears to encompass both immobility (which inhibits visual detection) and posture (which inhibits ingestion by gape-limited predators). However, further research is required to fully understand these two aspects.
A similar pattern of decreasing predator avoidance response with greater body size has been demonstrated in other marine invertebrates. For example, the American lobster Homarus americanus exhibits ontogenetic changes in its preferred microhabitats, preferring sheltering habitats early in life but expanding to an increasingly wide range of habitats as it grows (Wahle, Reference Wahle1992). Among Idotea, interference competition for microhabitats is influenced strongly by body size, with small males more likely than large males to leave a crowded habitat (Franke et al., Reference Franke, Gutow and Janke2007). If small males are forced to use habitats that are not protected from predators, they may exhibit a longer duration of tonic immobility to cope with the heightened predation risk. Nevertheless, this specific variation in habitat use has not been documented in C. isopus, and individuals in this study were all collected from the same habitat (under rocks) during low tide. However, it remains possible that microhabitat usage in this species differs by body size or sex during other tidal or diurnal periods.
Populations of C. isopus comprise individuals of various ages, reflecting the species’ lifespan of 13–15 months and its three synchronized reproductive cycles (Takahashi and Goshima, Reference Takahashi and Goshima2012). Thus, if the large individuals in our study were older than the small ones, our result may partially reflect an ontogenetic shift toward shorter tonic immobility in C. isopus. Larger individuals are at a more advanced developmental stage, and in males, there is a greater tendency to initiate precopulatory guarding during the breeding season (Jormalainen and Merilaita, Reference Jormalainen and Merilaita1995; Miura and Goshima, Reference Miura and Goshima2016), which may result in a decreased response to predation risk. During the breeding season, I. baltica males exhibit increased activity and have been documented to appear more reckless to predators (Jormalainen and Tuomi, Reference Jormalainen and Tuomi1989; Vesakoski et al., Reference Vesakoski, Merilaita and Jormalainen2008).
The lack of a discernible relationship between the duration of tonic immobility and body size in females might be attributed to a temporary behavioural restriction in ovigerous females. As in other isopods, C. isopus females incubate their embryos in the ventral brood pouch, and all females in our study had brood pouches containing embryos in an early stage of development (Takahashi and Goshima, Reference Takahashi and Goshima2012). In the terrestrial isopod Armadillidium vulgare, rolling behaviour is an effective anti-predator defence to protect the vulnerable ventral part, but ovigerous females have difficulty maintaining a rolled-up posture due to the presence of the brood pouch (Suzuki and Futami, Reference Suzuki and Futami2018). Ovigerous females might also have difficulties exhibiting tonic immobility due to the mechanical constraints of the brood pouch. Females that hold developed embryos in the brood pouch may instead rely on primary defences such as reducing activity to avoid detection by predators (Jormalainen and Tuomi, Reference Jormalainen and Tuomi1989). However, the lack of variation in the reproductive status and developmental stage of the embryos in our data hinders a comprehensive evaluation of this hypothesis.
There was no significant effect of colour morph on tonic immobility in our study. Tonic immobility often functions as a secondary defence, meaning it occurs after detection by predators (Endler, Reference Endler, Federand and Lauder1986). This could explain the lack of a strong relationship with colour morph, which is likely to function as a primary defence (i.e. it works prior to detection by predators). Alternatively, the lack of effect of colour morph could simply reflect the artificial conditions and uniform background colours used in our behavioural trials. Further studies on natural substrata that allow for effective concealment of each morph may reveal an interaction between colour morph and the duration of tonic immobility.
Our results partially explain variations in tonic immobility in the marine isopod C. isopus. However, our data do not demonstrate whether this behaviour is effective for avoiding predation and are not sufficient to identify which types of predators induce it. The effectiveness of anti-predator defences can depend on the relative speed of movement of predators and prey (Aguilera et al., Reference Aguilera, Weiß and Thiel2019). Tonic immobility might be advantageous for prey species that are extremely slow-moving in comparison to predators, meaning that fleeing is ineffective. Numerous studies in well-studied artificial selection systems have reported a negative relationship between locomotor performance and selection for tonic immobility (Ohno and Miyatake, Reference Ohno and Miyatake2007; Miyatake et al., Reference Miyatake, Okada and Harano2008; Nakayama et al., Reference Nakayama, Nishi and Miyatake2010, Reference Nakayama, Sasaki, Matsumura, Lewis and Miyatake2012; Matsumura et al., Reference Matsumura, Sasaki and Miyatake2016). Hazlett et al. (Reference Hazlett, Bach, McLay and Thacker2000) studied predator avoidance among five sympatric decapod species and documented immobile behaviour resembling tonic immobility in two of the less mobile species. Further studies are needed to determine whether this pattern applies more widely. Tonic immobility has also been observed in other Idoteidae, e.g. C. strasseni and I. ochotensis (K. Igarashi, personal observation). Understanding the taxonomic prevalence of tonic immobility and the predators that induce it will provide a general understanding of this behavioural defence in marine crustaceans. Some marine invertebrates (e.g. hermit crabs or gastropods) take refuge in shells and remain quiescent for several minutes. Although this behaviour resembles tonic immobility in some respects, it may be best to classify this as a distinct behaviour.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315424000134.
Data availability
The datasets for this study are available from the corresponding author upon reasonable request.
Acknowledgements
We thank the members of the Laboratory of Animal Ecology, Graduate School of Fisheries Sciences, Hokkaido University, for their advice and for their cooperation with the sampling and experiments.
Authors’ contributions
Koichi Igarashi: Conceptualization, Methodology, Formal analysis, Investigation, Writing–original draft, Writing–review & editing, Visualization. Satoshi Wada: Writing–original draft, Writing–review & editing, Supervision.
Financial support
This research received no specific grant from any funding agency, or from commercial or not-for-profit sectors.
Competing interests
None.
Ethical standards
The experiments conducted as part of this study, which involved crustaceans, did not require approval from the Hokkaido University Animal Experiment Committee, as per their guidelines. Consequently, no specific approval number was assigned. However, we made concerted efforts to minimize the distress experienced by the study animals throughout the course of the experiments.
