Does the millipede, Orthoporus texicolens, (Spirostreptida: Spirostreptidae) exhibit habituation to tactile stimuli? Isabella Newingham and Dr. Marianne Robertson Millikin Biology Department, Decatur, IL, 62522

• No significant main effect of trial independent of the group in Abstract Methods 23.5 and 21.5Results newton experimental Cont’d groups (P = 0.527). Discussion Response rate • We based our methodology on Bullock et al. (2009). We • No significant changes in response rate over time within • Throughout all trials, control and experimental millipedes North American millipedes, Orthoporus texicolens, coil as a housed O. texicolens in containers (11.0 cm diameter x 7.5 cm groups for both the 23.5 newton (P = 0.800) and 21.5 newton (P continued to coil in response to the tactile stimulus. defense mechanism when exposed to predators. We height) with 250 g of non-treated soil. We sprayed the soil with = 0.980) treatments. Additionally, there was no significant decrease in time O. 20 mL of tap water weekly to maintain a moist environment examined habituation, a type of non-associative learning texicolens spent coiled over subsequent trials at any pressure (Srinivasa and Mohanraju, 2011) and we provided the (25.0, 23.5, 21.5 newtons). However, in all trials no millipedes described as a diminished response to a repeated neutral millipedes with carrots and apples Ad libitum. stimulus, to tactile stimuli. We observed coiling in response to responded to 20.0 newtons. 25.0, 23.5, 21.5 and 20.0 newtons of force. Control and • We conducted preliminary observations to determine the • Therefore, our hypothesis that O. texicolens would habituate experimental groups differed in interval length between baseline behavior of the millipedes. To apply tactile stimuli, we to a 25.0, 23.5, and 21.5 newton tactile stimulus was not repetition of stimuli. We recorded presence versus absence of attached a spring scale to a ring stand and lowered it to touch each millipede. supported. However, our hypothesis that O. texicolens would coiling to the stimulus and the duration of each coiling not habituate to a 20.0 newton tactile stimulus was supported. response. There was no evidence of habituation at any • For the 25.0 newton treatment, we randomized millipedes into pressure; millipedes continued to coil in all trials. There was control (n = 18) and experimental (n = 18) groups. We exposed • These results were unexpected since O. texicolens does no significant difference in the length of response over trials control millipedes to the tactile stimulus twice for 10 sec, habituate to other stimuli (Bullock et al., 2009). in control or experimental treatments. However, there was an separated by an interval of 40 minutes. We exposed increase in the number of non-responses to the stimuli as experimental millipedes to the same tactile stimulus for 10 Figure 1. Presence or absence of response of millipedes, O. texicolens, after • Human error could have been corrected using an automatic pressure decreased. This demonstrates that it is adaptive for seconds over the course of 10 trials separated by 2-minute subjected to a tactile stimulus of 25.0 newtons in 246 trials. timing system rather than a stopwatch. this species to respond to pressure stimuli. intervals. • Pill millipedes responded differently to treatment groups of • For the 23.5 and 21.5 newton treatments, using a new sample touching, squeezing and dropping, which simulated different of millipedes, we exposed control millipedes (n = 12) and sizes of vertebrate predators, such as mammals, birds, and Introduction experimental millipedes (n = 12) to the same tactile stimuli reptiles (Tuf et al., 2016). procedure as the 25.0 newton treatment. • Millipedes are arthropods in class Diplopoda found in • O. texicolens may not habituate to a tactile stimulus as it is • For the 20.0 newton treatment, we exposed 48 millipedes to the forests and woodlands (Liu et al., 2017). not an innocuous stimuli (Hackl and Robertson, 2019) and the tactile stimulus for 10 seconds for one trial. potential threat of predation is greater than the reward of obtaining mates, food, etc. • Coiling can occur when millipedes feel threatened by • We recorded presence versus absence of a coiling to the tactile stimulus, the time it required to coil, the time millipedes predators or are stimulated by touch (Smigel and Gibbs, • There is a cost-benefit tradeoff between evolutionary benefits 2008). remained coiled, and the interval between each stimulus. Figure 2. Presence or absence of response of millipedes, O. texicolens, and costs of behaviors performed including foraging (Hassall after subjected to a tactile stimulus using 21.5 newtons and 23.5 newtons of and Tuck, 2007), mating (Rowe et al., 1994), and predation • Habituation is a type of non-associative learning Statistical tests: force in 288 trials. • Generalized linear model- proportion of responders and non- (Rowe et al., 1994). The coiling response of millipedes has described as a behavioral response that gradually responder fitness benefits to reduce risk of mortality but requires fitness reduces upon repeated innocuous stimulation (Frost et s costs such as reproduction and energy usage (Hassall and • Repeated measures ANOVA and paired t-test- length of time al., 2006; Nagayama and Araki, 2015). coiled Tuck, 2007). • Friedman’s test- response rate (presence versus absence of • Habituation research has been conducted on honeybees response) • Learning behavior, such as habituation, can influence (Baracchi et al., 2017), millipedes (Bullock et al., 2009) individual fitness, reproductive success and evolutionary and isopods (Hackl and Robertson, 2019). Results changes (Hackl and Robertson, 2019). • Dishabituation is a decreased response that has Figure 3. Average duration of coiling response of control millipedes (subjected to 25.0 Presence vs absence of response: newtons of force for 10 seconds separated by 40-minute intervals) and experimental Literature Cited undergone habituation as a result of a novel stimulus • In 246 trials, millipedes in two trials did not respond while millipedes (subjected to 25.0 newtons of force for 10 seconds separated by 2-minute (Bullock et al., 2009). millipedes in 244 trials responded to the 25.0 newton tactile intervals over 10 trials). stimuli (Fig. 1). Therefore, there was not enough variation of • Bullock et al. (2009) demonstrated that 75% of millipedes, reactions to stimuli to calculate a valid statistic. Orthoporus texicolens, habituated to a vibrational • In 144 trials, millipedes in eight trials did not respond while stimulus and of those who habituated, 100% of them millipedes in 136 of the trials did respond to the 23.5 newton dishabituated. tactile stimuli. In 144 trials, millipedes in 46 trials did not respond while millipedes in 98 trials did respond to the 21.5 • When exposed to a heat stimulus the isopod newton tactile stimuli (Fig. 2). • No significant group interaction between treatment group (23.5 Armadillidium vulgare showed a significant decrease in and 21.5 newtons) and interval (control/treatment) (P = 0.814). coiling after 20 trials (Hackl and Robertson, 2019). There was a significantly lower response rate between 23.5 and 21.5 newton treatment (P < 0.001), with a significantly lower Figure 4. Average duration of coiling response of control millipedes (subjected to • The purpose of this study was to determine whether O. proportion of responders in the 21.5 newton treatment, 23.5 newtons of force for 10 seconds separated by 40-minute intervals) and experimental millipedes (subjected to 23.5 newtons of force for 10 seconds separated texicolens demonstrates habituation to tactile stimuli of independent of treatment or interval. by 2-minute intervals over 10 trials). various pressures. • In 48 trials, all millipedes did not respond to the 20.0 newton stimuli. • We hypothesized O. texicolens would habituate to a Length of time coiled: tactile stimulus of 25.0 newtons, 23.5 newtons, and 21.5 • No significant difference in the length of time O. texicolens newtons. However, we hypothesized O. texicolens would remained coiled after being exposed to a 25.0 newton tactile not experience habituation in response to a tactile stimulus in the control treatment (P = 0.986) or the Acknowledgments stimulus of 20.0 newtons. experimental treatment (P = 0.223) (Fig. 3). • No significant interaction term between pressure group and time in 23.5 and 21.5 newton control groups (P = 0.140). • When the organism receives environmental cues, it is • No significant difference in length of time coiled from trial 1 to We thank Dr. Travis Wilcoxen for help with data analysis, necessary to react to harmful stimuli, such as predation Figure 5. Average duration of coiling response of control millipedes (subjected to trial 2 in 23.5 and 21.5 newton control groups (P = 0.761). 21.5 newtons of force for 10 seconds separated by 40-minute intervals) and Sue James for help with ordering supplies, and the Millikin (Bullock et al., 2009), while learning to ignore innocuous • No significant interaction between group and trial in 23.5 and experimental millipedes (subjected to 21.5 newtons of force for 10 seconds Biology department for funding our research. stimuli to conserve energy (Frost et al., 2006). 21.5 newton experimental groups (P = 0.360). separated by 2-minute intervals over 10 trials).