Subjects and Housing
In total, 44 adult female praying mantises (Tenodera aridifolia) were used as predators. Males were excluded because of their variable foraging behavior and overall lower level of prey ingestion (e.g. Carle et al. 2015), compared to females. Oothecae were collected in a suburb of Fukuoka (Japan) on grassland near Tachibana mountain (+33° 40′ 46.7′′, +130° 28′ 6.20′′). The nymphs obtained were bred to adulthood using methods previously described (e.g. Sato and Yamawaki 2014; Carle et al. 2015). The mantises were kept at 25 ± 3 °C and in a 12 h:12 h L:D photoperiod (light phase, 9:00–21:00) during breeding. They were kept together in a plastic box (40 × 23 × 25 cm) provided with mesh walls inside for moulting and with aerations on the top. During breeding, they were fed with fruit flies (Drosophila melanogaster) three times per week, in addition to water ad libitum until the 3rd instar. Then, we provided nymphs of crickets (Acheta domesticus, ca. 5–20 mm lengths) at the same frequency. At this moment, individuals were isolated and placed in similar plastic boxes that were partitioned into nine compartments (13 × 7 × 25 cm), and water was sprayed at the top of cages after food was provided. At adulthood, each individual was placed in an individual box (15 × 10 × 20 cm) and received the same diet as previously. Prior to experiments, the mantises were food deprived for 3 days.
Prey
Three different prey species were used: crickets (Acheta domesticus, ca. 10 mm in length) as familiar prey to the mantis, mealworms (Tenebrio molitor larvae, ca. 20 mm in length) as novel and relatively cryptic prey, and honeybee workers (Apis mellifera, 10 mm in length: their sting and associated venom gland were removed to avoid the natural toxins of bees) as conspicuously novel prey. The mealworms were chosen based on our previous work showing that the mantises stop eating mealworms injected with 500 mM of DB (Carle et al. 2015), and because they are less conspicuous than bees, to the mantis visual system (see below). All of these prey species were obtained from commercial suppliers. Each mantis only received two of these prey species as follow: cricket/worm, bee/worm or cricket/bee.
Contrast of Prey
The visual (Michelson) contrast of the prey, as seen through the mantis’s compound eye, was calculated as:
$$ contrast=\left({\mathrm{q}}_{prey}-{\mathrm{q}}_{board}\right)/\left({\mathrm{q}}_{prey}+{\mathrm{q}}_{board}\right) $$
where q
prey
is the response of the photoreceptor in the mantis compound eye to the light reflected from the prey, under the experimental conditions, and q
board
is the response to the whiteboard against which the prey is displayed. These responses are calculated using standard models for surface-light interactions and receptor activations (e.g. Wandell 1995; Hurlbert 1998; Kinoshita and Arikawa 2000; Fabricant and Herberstein 2014):
$$ {\displaystyle \begin{array}{l}{\mathrm{q}}_{prey}={\int}_{\lambda}\mathrm{s}\left(\uplambda \right){I}_{prey}\left(\uplambda \right)d\uplambda \\ {}{\mathrm{q}}_{board}={\int}_{\lambda}\mathrm{s}\left(\uplambda \right){I}_{board}\left(\uplambda \right)d\uplambda \end{array}} $$
where
$$ {I}_{prey}\left(\uplambda \right)={\mathrm{r}}_{prey}\left(\uplambda \right)E\left(\uplambda \right) $$
$$ {I}_{board}\left(\uplambda \right)={\mathrm{r}}_{board}\left(\uplambda \right)E\left(\uplambda \right) $$
and r
prey
(λ) corresponds to the surface spectral reflectance of the prey’s body; r
board
(λ) the surface spectral reflectance of the whiteboard; E(λ) the spectral power distribution of the illumination; I
prey
(λ) the surface spectral radiance reflected from the prey’s body (and similarly for the whiteboard). For the spectral sensitivity of the mantis visual receptors, s(λ), we used the sensitivity of the dark-adapted compound eye in mantis Tenodera sinensis, as provided in Sontag (1971) (Fig. 1).
Surface radiance measurements were made from a 0.2 degree spot centred on the prey’s body (for the bee, the spot was located on the darkest portion of the bee’s body), from a distance of approximately 50 cm (i.e. a spot less than 2 mm in diameter on the prey’s body), using a Konica Minolta CS-2000 spectroradiometer, under (1) the experimental illumination (positioned as in the experiment, at a distance of 50 cm above the board) and (2) a broad-band white light. Surface radiance measurements were made from the experimental whiteboard directly adjacent to the prey, under the same illumination conditions. Analogous measurements and definitions apply for both mealworm and bee.
The computed visual (Michelson) contrasts are −0.81 for the bee and −0.50 for the mealworm. Both contrasts are negative, i.e. the prey are darker than the background, and fall in the tracking and striking range (Prete et al. 2011), but the visual contrast of the bee is 1.63 times greater than that of the mealworm.
Experimental Set-up
During the experiments, the mantises were tethered to an apparatus (Fig. 2a). A piece of pin header (2131D2*40GSE, Linkman, Japan) was stuck on the dorsal pterothorax of the mantises with beeswax, and a piece of pin socket (21602x40GSE, Linkman, Japan) was fixed on the terminal tip of a flexible arm. During the experiments, the mantises were tethered to the arm by inserting the pin header into the socket, and were positioned on a Styrofoam ball (12 cm in diameter) that was kept airborne by a small fan, allowing them leg movements. They were then given one hour to acclimate to the apparatus before starting the protocols.
During the protocols, because it has been shown that speed may affect mantises’ decision-making for attacking (e.g. Prete et al. 1993), the prey were mechanically moved leftward or rightward at a constant speed of 205 mm/s, by a custom-made apparatus modified from Yamawaki (2011), as this speed successfully elicit strikes in mantises (preliminary experiments). The prey was impaled with a needle onto a platform. After being impaled, we did not observe movements of the prey as we smashed the content of their abdomen and thorax with a needle before injecting them (see below). The platform held two needles, the one for normal prey and the other for bitter prey (Fig. 2b), in order to avoid any taste contaminations. Only one of these needles was used in a given presentation, and these needles were separated from each other (140 mm) to avoid the disturbance by the unused needle. The platform was horizontally moved along rails by an electronic motor (US206–401, Oriental Motor) and pulleys. The distance to prey was kept at 2 cm when the prey was located in front of the mantis, which was the optimal distance for the tethered mantises to capture the prey in our preliminary experiments. Note that although the three prey types differ in size and therefore subtend different viewing angles at the mantis eye, all are well above the size threshold for eliciting the maximum tracking and striking behavior (Prete et al. 2011) (e.g. the bee and mealworm subtends approximately 30 and 90 degrees of visual angle in length and 10 and 5 degrees in width, respectively, when located at 20 mm from the centre of the mantis’s view). The vertical position of the prey was adjusted to the centre of the mantis’s head. The mantises were surrounded with white walls in order to prevent any visual distraction, and their behaviour was monitored using a video camera (HDR-XR520V, SONY) placed above the apparatus.
Protocol
The protocol consisted of an acclimation session (during 3 days) followed by one day off and a learning session (during 6 days). In a session, the mantises received a trial per day that consisted of three presentations for each of two prey species (six presentations in total) with an interval of 30 min between each prey (see Fig. 3), as the female mantises showed that they are able to attack and eat about 8 mealworms per day (Carle et al. 2015). The presentation order was randomly determined for each individual. A presentation consisted of mechanically moving the prey in front of a mantis, waiting 10 s for an attack, and then moving the prey away. This sequence of prey movements was repeated four times during each presentation, until the mantis attacked the prey. For each trial, we measured the numbers of prey attacked and eaten.
During the acclimation session, both of the two prey species were injected in their abdomen and thorax with and coated by 100 μl of distilled water to ensure that the prey have the same shape after injection and no movements (due to our method of injection) as during the learning phase. The purpose of this session was to ensure that the mantises acclimated to the apparatus, and to investigate the preference of mantises for prey species. During the learning session, the prey were injected with a 100 μl solution of either 500 mM denatonium benzoate (Tokyo Chemical Industry, Japan) or distilled water. The concentration of denatonium benzoate (DB) was chosen based on our previous work showing that female mantises show aversive behaviour at this concentration (Carle et al. 2015). To examine the effects of prey type on avoidance learning, two different treatments (combinations of prey species and solutions) were used. For example, in the cricket/worm condition, half of mantises received crickets injected with water and mealworms injected with DB, and the other half of mantises received the reverse treatment (DB-injected crickets and water-injected mealworms).
Data Analysis and Statistics
The data were plotted in excel files (Microsoft office, Microsoft corporation) and the statistical analysis was carried out using SPSS version 22 (IBM Corporation, www.ibm.com/software/analytics/spss). Because the data were not normally distributed (Shapiro-Wilk tests: P < 0.05), we employed generalized estimating equations (GEEs) that allowed us to use a more appropriate distribution to analyse these data (e.g. Poisson distribution with identity link function). We also reduced the risk of familywise errors by applying Holm-Bonferroni corrections.
First, we checked the response to the type of prey depending on the other prey presented in a pair (for example, we checked if there were significant differences in responses to crickets between the cricket/worm and cricket/bee conditions). Because we did not find any effects of the other prey (GEEs; for all values, χ22 < 1.89, P > 0.05) or any interaction with another factor (for all values, χ21–2 < 4.88, P > 0.05), we pooled the data together for the main analyses and provide the detailed experiments as supplementary files. Therefore, for the acclimation session data, we employed GEEs with types of prey (crickets, bees or mealworms) as categorical factor and days as ordinal factor, and adjusted α depending on Holm-Bonferroni corrections. For those of the learning session, we used GEEs using prey, days and bitterness (DB or water) as factors, and adjusted α to 0.0166 for the factor inducing the most significant result, to 0.025 for the factor inducing the second most significant result, and to 0.05 for the last factor.
Ethical Notes
Because T. aridifolia is neither an endangered nor a protected species in Japan, no specific permission was required for collecting the oothecae. Moreover, our experimental procedure did not involve any physical damage. Through all the experiments, in total 13.6% of mantises (N = 6) died. However, because the mantises were freely able to ingest or reject prey and that bitter prey were easily detectable because they were coated with bitter solutions, it is unlikely that these deaths were due to bitter compounds ingested. During the manipulations, we took care to gently handle the mantises when we moved them between their cage and the experimental set-up. Furthermore, we anesthetized the mantises with cold, before fixing a piece of pin header. After these experiments, the mantises were used for physiological studies.