Beetle bombing always deters praying mantises

Some animals have evolved chemical weapons to deter predators. Bombardier beetles (Coleoptera: Carabidae: Brachininae: Brachinini) can eject toxic chemicals at temperatures of 100 °C from the tips of their abdomens, ‘bombing’ the attackers. Although some bombardier beetles can reportedly deter predators, few studies have tested whether bombing is essential for successful defence. Praying mantises (Mantodea) are ambush predators that attack various arthropods. However, it is unclear whether bombardier beetles deter mantises. To test the defensive function of bombing against praying mantises, I observed three mantis species, Tenodera sinensis, Tenodera angustipennis, and Hierodula patellifera (Mantidae), attacking the bombardier beetle Pheropsophus jessoensis (Carabidae: Brachininae: Brachinini) under laboratory conditions. All mantises easily caught the beetles using their raptorial forelegs, but released them immediately after being bombed. All of the counterattacked mantises were observed to groom the body parts sprayed with hot chemicals after releasing the beetles. When treated P. jessoensis that were unable to eject hot chemicals were provided, all mantises successfully caught and devoured the treated beetles. Therefore, bombing is essential for the successful defence of P. jessoensis against praying mantises. Consequently, P. jessoensis can always deter mantises.


Study organisms
I collected 60 adult P. jessoensis from grasslands and forest edges in the Kinki region (Hyogo and Shiga Prefectures) of Japan, in May 2018, May-September 2019, and July-September 2020 (cf. Sugiura & Sato, 2018;Sugiura, 2018). The beetles were kept individually in plastic cases (diameter 85 mm; height 25 mm) with wet tissue paper under laboratory conditions (25 ± 1 C; Sugiura & Sato, 2018;Sugiura, 2018). Dead Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae were provided as food (Sugiura & Sato, 2018;Sugiura, 2018). Before the experiments, I weighed the beetles to the closest 0.1 mg using an electronic balance (PA64JP; Ohaus, Tokyo, Japan) and measured the body length to the closest 0.01 mm using slide callipers. Beetles were not used repeatedly in different feeding experiments. I conducted the following experiments 57.1 ± 5.0 (range: 5-162) days after collecting the beetles.
Mantises were kept individually in plastic cases (diameter 100 mm; height 100 mm) with wet tissue paper under laboratory conditions (25 ± 1 C). Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) and wild-caught insects (e.g., grasshoppers) were provided as food. The mantises were starved for 24 h before the feeding experiments to standardise their hunger level (cf. Sugiura, 2018). I weighed them to the closest 0.1 mg using an electronic balance (PA64JP; Ohaus, Tokyo, Japan) and measured the body length to the closest 0.01 mm using slide callipers. As with the bombardier beetles, individual mantises were not used repeatedly. I conducted the following experiments 11.3 ± 1.5 (range 1-45) days after I collected the mantises.

Experiments
To test the effectiveness of the anti-predator defences of P. jessoensis against praying mantises, I conducted the behavioural experiments under laboratory conditions (25 ± 1 C).
First, I placed an adult mantis on a plastic net in a transparent plastic case (length × width × height, 120 × 85 × 130 mm), so that the mantis hung its head down below its legs ( Fig. 1). Then, I placed a live P. jessoensis ('control' beetle) on the bottom of the case. Mantises that did not respond to the beetle were not used for the experiments. When a mantis displayed attacking behaviour (i.e., shooting out its forelegs to capture the prey), I recorded the behaviour on video using a digital camera (iPhone XS; Apple Inc., Cupertino, CA, USA) at 240 frames per second. If the mantis rejected the beetle after attacking it, I observed whether the mantis reattacked the same beetle within 1 min. Rejected beetles were also checked for injuries. If a mantis started to eat the beetle, I recorded the feeding time. I also weighed any uneaten beetle parts and calculated the percentage of the beetle eaten. In total, 30 control beetles and 30 mantises (10 T. sinensis, 10 T. angustipennis, and 10 H. patellifera) were used in the experiments.
To test whether the bombing response of P. jessoensis plays an essential role in deterring a mantis, I provided the mantises with treated P. jessoensis that were unable to eject hot chemicals. Following the method of Sugiura & Sato (2018), I repeatedly stimulated an adult P. jessoensis with forceps; the simulated attacks forced them to exhaust their chemicals (i.e., 'treated' beetles). Then, I observed whether an adult mantis successfully attacked the treated beetle in a transparent plastic case (length × width × height, 120 × 85 × 130 mm) using the same procedure as for the control beetles. In total, 30 treated beetles and 30 mantises (10 T. sinensis, 10 T. angustipennis, and 10 H. patellifera) were used in the experiments.
All experiments were performed in accordance with Kobe University Animal Experimentation Regulations (Kobe University Animal Care and Use Committee, No. 30-01). Data analysis I used Fisher's exact test to compare reattack rates between mantis males and females and successful escape rates between control and treated P. jessoensis from each mantis species and all mantis species combined. I used Student's t-test to compare the body sizes of P. jessoensis and mantises between the control and treatment experiments. All analyses were conducted using R ver. 3.5.2 (R Core Team, 2018).

RESULTS
All mantises used their raptorial forelegs to capture P. jessoensis. However, all of the control beetles ejected hot chemicals immediately after being captured and the mantises released the beetles immediately after being bombed (n = 30; Figs. 2 and 3; Video S2). The chemicals ejected by P. jessoensis were sprayed on the head, forelegs, and/or thorax of each mantis. In T. sinensis, 60% of females (n = 5) and 20% of males (n = 5) reattacked P. jessoensis within 1 min after releasing them. In T. angustipennis, 33.3% females (n = 6) and 0% of males (n = 4) reattacked P. jessoensis. In H. patellifera, 20% of females (n = 10) reattacked P. jessoensis. Mantis females reattacked P. jessoensis more frequently than mantis males; however, these differences were not significant (Fisher's exact test; T. sinensis, All reattacking mantises rejected P. jessoensis again after being bombed. No mantis successfully preyed on control beetles (Fig. 2). After releasing the beetles, all of the mantises were observed to groom the body parts sprayed with hot chemicals. No released P. jessoensis was injured; all were active (n = 30). When treated P. jessoensis that were unable to eject hot chemicals were provided, all the mantises successfully caught the treated beetles using their raptorial forelegs (Video S3). All of the mantises devoured the treated beetles (n = 30; Figs. 2 and 4; Video S3). The mantises consumed 90.5% of the body (mainly the thorax and abdomen) of treated P. jessoensis, while parts of the elytra, legs, and antennae were not eaten (Table 1; Fig. 4). The mean ± standard error feeding time was 52.3 ± 6.6 min ( Table 1).
The rates of successful escape from mantises significantly differed between the control and treated P. jessoensis (Fisher's exact test; T. sinensis, P < 0.0001; T. angustipennis, P < 0.0001; H. patellifera, P < 0.0001; all species combined, P < 0.0001). The mean body sizes (lengths and weights) of mantises that attacked control and treated beetles did not differ significantly ( Table 2). The mean body sizes (lengths and weights) of control and treated beetles did not differ significantly (Table 2). Therefore, the bombing responses of adult P. jessoensis deterred mantises of all species, sexes, and sizes.

DISUSSION
Some praying mantises can prey on well-defended insects, although the predation success rate varies among prey insect species (Reitze & Nentwig, 1991). The effectiveness of chemical defences by bombardier beetles against mantises remains unclear (Eisner, 1958). In this study, I tested the effectiveness of the defences of the bombardier beetle, P. jessoensis, against three mantis species under laboratory conditions (Fig. 2). My  experiments demonstrated that bombing was essential for successful defence by P. jessoensis against mantises, which were always deterred (Fig. 2). To my knowledge, this is the first study to document a perfect defence against praying mantises by insects smaller than mantises. Dean (1980b) experimentally investigated the relative importance of the toxic chemicals and heat produced by bombing for the successful defence of bombardier beetles against toads. Although the combination of chemicals and heat played an important role in deterring toads, the chemicals served as the primary defence and bombing as a secondary defence (Dean, 1980b). Toxic chemicals or other material on the body of P. jessoensis functioned as a primary deterrent against frogs (Sugiura, 2018) and birds (Kojima & Yamamoto, 2020), suggesting that bombing is not essential for the successful defence of P. jessoensis against frogs and birds. However, all praying mantises consumed the treated P. jessoensis (Fig. 2), suggesting that chemicals on the body of P. jessoensis could not deter mantises. Studies have indicated that chemically defended arthropods could not effectively deter mantises (Reitze & Nentwig, 1991). For example, mantises such as T. sinensis could consume toxic caterpillars after removing ('gutting') the midgut containing toxic plant material (Rafter, Agrawal & Preisser, 2013;Mebs et al., 2017;Mebs, Wunder & Toennes, 2019). Several mantis species could also tolerate noxious chemicals such as tetrodotoxin, cardenolides, and quinine used as anti-predator defences by toxic arthropods (Mebs, Yotsu-Yamashita & Arakawa, 2016;Mebs et al., 2017;Rafter et al., 2017aRafter et al., , 2017bMebs, Wunder & Toennes, 2019). Therefore, bombing plays an essential role in defending against mantis predation, although additional experiments are needed to test the importance of heat in the successful defence of P. jessoensis against mantises.
Some predators avoid attacking bombardier beetles after experiencing the toxic chemicals (Dean, 1980a;Kojima & Yamamoto, 2020). Dean (1980a) found that many American toads, Anaxyrus americanus (Holbrook) (Anura: Bufonidae), did not reattack bombardier beetles (Brachinus spp.) for at least 30 min after rejecting them. Kojima & Yamamoto (2020) observed that some quail exposed to live P. jessoensis avoided them for up to 5 weeks. In this study, 26.7% of mantises reattacked P. jessoensis within 1 min after being bombed; P. jessoensis mantises were reattacked more frequently by females than by males, although these differences were not significant. Hungrier mantises (starved >24 h) may be more likely to reattack P. jessoensis after being bombed. However, P. jessoensis should be capable of easy escape from mantises before they reattack under field conditions, because P. jessoensis can rapidly leave the site after release.
Chemically defended prey produce toxic chemicals that force predators to spit them out (Taniguchi et al., 2005;Whitman & Vincent, 2008;Matsubara & Sugiura, 2017). However, the first predator attack potentially damages the defended prey. Therefore, the chemically defended prey may have evolved tolerance for predator biting and other attacks (Sugiura & Sato, 2018;Sugiura, 2020a). In this study, none of the P. jessoensis released by mantises were injured, suggesting that P. jessoensis has a body tough enough to survive an attack by the raptorial forelegs of mantises.