Behavioural Processes 157 (2018) 422–430

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Behavioural Processes

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Aggregative behavior and intraspecific communication mediated by T substrate-borne vibrations in terrestrial : An exploratory study in two species of woodlice ⁎ Sofia Cividinia, Giuseppe Montesantob, a Independent Researcher in Biology and Biostatistics, Como 22100, Italy b Dipartimento di Biologia, Università degli Studi di Pisa, via A. Volta 4bis, Pisa 56126, Italy

ARTICLE INFO ABSTRACT

Keywords: Gregarious behavior and aggregative phenomena among conspecifics in woodlice are thought to be a form of Crustacea evolutionary adaptation to the terrestrial environment which has given these multiple advantages, e.g., Oniscidea against desiccation and predation. The reasons behind these phenomena, however, have not fully been clarified Armadillo officinalis yet. This exploratory study has the aim to assess displacement and aggregation state relatively to the presence of Aggregation substrate-borne vibrations in two different species of terrestrial isopods. To this goal, a circular arena was usedto Vibrational communication collect data from adult individuals of Armadillo officinalis, a stridulating species, and Armadillidium vulgare, a Gregarious behavior non-stridulating species, both exposed and non-exposed to micro-vibrations. Our results showed that exposed individuals of A. officinalis significantly react to micro-vibrations positioning themselves preferentially far from the vibrational source compared to non-exposed individuals of the same species. Furthermore, both the number of aggregates and of isolated subjects significantly increase in the presence of substrate-borne vibrations thanin the absence of micro-vibrations. No statistically significant association with substrate-borne vibrations was found for A. vulgare for both placement and number of aggregates and of isolated subjects. Unlike A. vulgare, A. officinalis appears very sensitive to micro-vibrations which seem to represent a source of disturbance or potential danger. Substrate-borne vibrations seem to affect the capability of this species to aggregate leading to agreater number of aggregates and isolated subjects as if animals were a bit disoriented. This behavior might be related to a possible capability of this species to use micro-vibrations for inter- and intraspecific communication.

1. Introduction evolved to provide these animals with multiple advantages besides preserving from dehydration. In peracarid , Thiel (2011) Aggregation is a well-known phenomenon in terrestrial isopods, reported that aggregative phenomena are used by these animals to although the reasons why it happens have not fully been clarified yet. It defend themselves from both predators and adverse environmental is thought to be mainly a form of evolutionary adaptation developed conditions. Possible chemical signals of recognition among conspecifics during the passage from the sea to the terrestrial environment to pre- such as aggregation pheromones might favor this phenomenon (Broly vent desiccation (Quinlan and Hadley, 1983). Terrestrial isopods can et al., 2012). fast dehydrate, losing water through the gills on pleopods, ventral part The goal of this exploratory study is thus to assess which other of pereion, and dorsal surface (Edney, 1951). Indeed, their cuticle is factors might be involved in favoring or disadvantaging gregarious equipped with many pores through which water may easily escape, and behaviors and aggregative phenomena in terrestrial isopods using the low content of lipids of the endocuticle further disadvantages water Armadillo officinalis, a stridulating species, as an experimental model in retention (Hadley and Quinlan, 1984). The close physical contact with comparison with Armadillidium vulgare, a species without stridulatory conspecifics might thus limit the body surface exposed to air reducing organ. water evaporation and preventing in this way desiccation (Edney, 1954, Armadillo officinalis Duméril, 1816 is a terrestrial isopod (Crustacea, 1968; Broly et al., 2014). Aggregation, however, might also represent a , Oniscidea) which belongs to the family Armadillidae and has mechanism of defense from predators (Broly et al., 2013) and be adapted to live in arid environments, predominantly, in the

⁎ Corresponding author. E-mail addresses: [email protected] (S. Cividini), [email protected] (G. Montesanto). https://doi.org/10.1016/j.beproc.2018.07.006 Received 22 May 2018; Received in revised form 16 July 2018; Accepted 16 July 2018 Available online 17 July 2018 0376-6357/ © 2018 Elsevier B.V. All rights reserved. S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430

Fig. 1. On the left side, the diagram of the arena used as a test apparatus with the position of the divisors; S, the position of the vibrational source; H, high vibrational intensity; M, medium vibrational intensity; L, low vibrational intensity. On the right side, the levels of acceleration (in m/s2) recorded during a 10-s test at the points indicated (gray circles) in the three corresponding sectors of the arena. The amplitude of the micro-vibrations, measured as root-mean-square amplitude (RMS vibration) of the vibration data about zero, decreases progressively from the sector H to the sector L.

Mediterranean basin and on western coasts of the Black Sea to prevent water loss and avoid the attack of predators (Svádová et al., (Schmalfuss, 1996, 2003). This species mainly has nocturnal habits 2014; Campbell and Stastny, 2015). (Vandel, 1962) and usually lives on sand, silty-clayey substrates, and Another salient characteristic of this species is that A. officinalis can rocks, as well as in environments populated by different plant com- emit vibrations utilizing a ledge of scales situated on the propodus of munities (Messina et al., 2011, 2012, 2014). The individuals of this the fourth and fifth pereopod (Caruso and Costa, 1976; Taiti et al., species are frequently found under rocks (or other shelters) where they 1998). Verhoeff (1908) had already described this feature, which is form large enough aggregates. This behavioral pattern has been ob- present in all species of the genus Armadillo (Schmalfuss, 1996). served in most of the terrestrial isopods and might have the double aim In a recent study on behavioral processes in woodlice, the authors

423 S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430 found a statistically significant association between the pattern of turn at the point indicated in Fig. 1 and used to produce micro-vibrations alternation and both exposure to substrate-borne vibrations and species comparable in intensity with those generated in a natural context. The of the animals (Cividini and Montesanto, 2018a). According to our best micro-vibration emission lasted for all time in which animals were in- model, A. officinalis not exposed, and A. vulgare exposed to micro-vi- side the arena. The segments with the acoustic recording needed to brations have 97 and 98% lower odds, respectively of being in a higher generate the micro-vibrations were produced with the software Auda- category of alternating turns compared to a lower category than ex- city (ver. 1.2.4). The utilized settings are indicated in Appendix A. posed individuals of A. officinalis. Armadillo officinalis seems to be very The audio file was carefully inspected, checked for the clipping, and reactive to substrate-borne vibrations, unlike A. vulgare, and this re- saved in a WAV digital format (16-bit amplitude resolution). The vi- activity might depend on the ability of this species to emit vibrations brational diagrams, in Fig. 1, show the levels of acceleration (in m/s2) (Cividini and Montesanto, 2018a). The authors also found evidence that recorded during a 10-s test in the three sectors of the arena at the level adults of A. officinalis can perform a significantly greater number of of the corresponding central points. In the same figure, the amplitude of alternating turns than exposed juveniles of the same species both in the the micro-vibrations, measured as the root-mean-square amplitude of absence (48% increase in the estimated mean) and in the presence the vibration data about zero (RMS vibration), was reported. The RMS (100% increase in the estimated mean) of substrate-borne vibrations parameter takes into account the vibration trend along time and gives a (Cividini and Montesanto, 2018b). This result seems to indicate a pro- value of amplitude directly related to the energetic content of vibration. gressive improvement in the turn alternation use, passing from the ju- The amplitude of micro-vibrations progressively decreases from the venile stage to the adult condition, as well as an increased reactivity to sector H to the sector L on all the three axes X, Y, and Z. substrate-borne vibrations. The software VIBSENSOR, running on Android 7.0 device (Huawei The capability of producing vibrations and stridulations by this P9, with the oscilloscope inside) was used to record the micro-vibra- species might also be related to aggregation phenomena, as a means of tions obtained from the emitted sound. intraspecific communication (Warburg and Berkovitz, 1978; Sibly, 1983; Hornung, 2011; Broly et al., 2012). 2.3. Procedure This study thus aims to evaluate if substrate-borne vibrations lead single individuals of A. officinalis, placed in a shared space, to modify Animals were individually put in the center of the arena by means of their reciprocal behavior at both the level of displacement and ag- a removable cylinder so that every was free to move in any gregation state. Based on the behavior observed in our previous studies, direction along with all the others. We left a two-hour interval to ani- A. officinalis seems to warn substrate-borne vibrations as a coming mals to acclimate and reach a stable balance, and we monitored their danger, or an adverse condition to avoid, increasing the number of turn displacements taking photos at 5, 30, and 60 min without flash for not alternations as a potential strategy of defense and escape. The high influencing their behavior (Fig. 2). We took the last photo at 120 min sensitivity to vibrations of this species might fit into a complex network after the release of the animals. After that, two dividers were used to of intra and interspecific signals of communication with both con- break down the arena into three predetermined sectors, namely upper specifics and the environment. The capability of producing stridulations (U), middle (M), and lower (L) having a decreasing gradient of vibra- might be involved too. Hence, to go in deep with this aspect, we set tional intensity when the device had switched on (Fig. 1). The animals ourselves the following goals. Our first goal is to evaluate whether inside each sector were so picked up and put into three different con- animals tend or not to position themselves as far away as possible from tainers opportunely named (U, M, and L). Hence, we counted the the zone with the highest vibrational intensity independently of their number of individuals per sector and determined gender and cepha- aggregation state. Our second goal is to assess whether, in the presence lothorax width (in mm) of each animal. The animals, which were part of substrate-borne vibrations, animals tend to form a lower or higher of an aggregate, but slightly trespassing, were put into the container number of aggregates, or if there is an increased number of isolated corresponding to the aggregate's sector. The 146 animals of each individuals. sample were then divided into smaller and larger sizes using the cor- responding median value of the cephalothorax width as the central 2. Materials and methods position index. The photos taken at 120 min from the release of animals were uploaded into the program ImageJ (ver. 1.51 r) to collect data 2.1. Subjects related to i) the number of aggregates per sector, ii) the number of animals inside each aggregate or alone, and iii) the distance in cm of Since April 2015 numerous specimens of A. officinalis and A. vulgare each animal from the source of micro-vibrations. We defined as ag- have been collected in Sicily (37°31′39″ N 15°04′20″ E) and bred in Pisa gregate at least two animals in contact. (43°43′07″ N 10°23′45″ E), in a climate room at 20 °C with a natural photoperiod. Animals were housed in a terrarium and fed with potato 2.4. Statistical analysis tubers and plane tree leaves (Montesanto and Cividini, 2017). In the present study, we used two random samples of 146 subjects each, of We defined the outcome variables as follows: i) an ordinal variable adult individuals of A. officinalis and A. vulgare. All the animals were representing the vibrational sectors where the displacement of the an- examined to check their bodily integrity before starting the experiment. imals occurred independently of being part of an aggregate, or not, and All the included animals were in intermolt or premolt phase ii) two count variables representing the total number of aggregates, and (Montesanto and Cividini, 2018). The individuals of each sample were the total number of isolated individuals per sector, respectively. The thus randomized to the control group (no micro-vibrations) or the ex- first variable was used to evaluate whether animals were attracted, or posure group (micro-vibrations) in a percentage of 50% per group (73 disturbed by substrate-borne vibrations positioning themselves conse- animals each) using a table of random numbers and a pseudorandom quently in a zone with a greater or lower vibrational intensity, re- sequence of 0s (control group) and 1s (exposure group). spectively (first goal). The other variables instead were used toassess the possible relationship between aggregation state and substrate-borne 2.2. Apparatus vibrations presence (second goal). We calculated the sample size for a medium effect size with For testing the behavior of animals, we used a circular arena of 34- G*Power 3.1 (Faul et al., 2009) to have a power equal to or greater than cm diameter made of high impact polystyrene (HIPS). 80% for the following statistical tests: chi-square test, t-test and Wil- A moving-coil miniature earphone, well hid from animals’ sight and coxon-Mann-Whitney nonparametric test for two independent samples, firmly fixed with hot glue, was located on the lower surface of thearena one-way ANOVA (α = 0.05). For the Fisher’s exact test, we considered

424 S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430

Fig. 2. Outline of the aggregation dynamics of Armadillo officinalis (AO) not exposed (a) and exposed (b) to micro-vibrations, after 5, 30, 60 and 120 min from the release of the animals in the center of the arena. The source of micro-vibrations is indicated in black (■) or white (□) when it is OFF or ON, respectively. the following proportions for animals in the sector H, in the absence individuals after two hours. The dynamics of A. vulgare were very si- and presence of micro-vibrations, respectively: p1 = 0.90 and milar in both absence (Fig. 3a) and presence (Fig. 3b) of micro-vibra- p2 = 0.70. Subsequently, the possible associations between the place- tions despite in the first 5 min, there was less dispersion for the exposed ment of the animals inside the test apparatus and the substrate-borne group. vibrations were studied more thoroughly with ordinal logistic regres- The Fisher’s exact test carried out on the individuals of A. officinalis sion models. For the parameter estimates, we used the generalized es- independently of their aggregation state showed that there is a statis- timating equations (GEEs) methodology, introduced by Liang and Zeger tically significant relationship between micro-vibrations and the sector (1986), to take into account the possible correlation among observa- where animals locate themselves (p < .0001). tions. A dichotomous variable representing the exposure state of the The possible association between the placement of the individuals of animals was used in the regression models. Gender and size were A. officinalis inside the test apparatus and substrate-borne vibrations considered as potential confounders and included in the models as was thus investigated more thoroughly fitting ordinal logistic regres- binary variables. sion models with and without interactions. The models with interac- Any possible relationships between i) the number of aggregates, or tions were not considered because the interaction terms were all sta- ii) the number of isolated individuals per sector and substrate-borne tistically non-significant. The models without interactions showed very vibrations were investigated with Poisson regression with robust stan- similar values of the fit statistics for the comparison of nested models dard errors, and with generalized distribution. (QIC). Hence, we chose the full model because biologically more in- Data analysis was carried out with SAS 9.4 by using the following formative than the reduced models. Table 2 reports the results of the procedures: proc freq, proc genmod, proc fmm, proc glm, proc npar1way, full model. The model found a statistically significant association be- and other standard procedures for descriptive statistics. The graphs tween the placement of the animals inside the test apparatus and micro- were created with the ggplot2 library (Wickham, 2009) in R/RStudio vibrations. According to our best model, thus, we can draw the fol- (ver. 3.4.3). lowing conclusions. Individuals of A. officinalis exposed to micro-vi- The diagrams and plates in Figs. 1–3 were drawn and/or arranged brations have 98% lower odds of being in a higher category of vibra- using the GNU Image Manipulation Program (GIMP) (ver. 2.8.22) with tional intensity versus a lower category than non-exposed individuals of the methods described in Montesanto (2015, 2016). the same species, holding all other variables constant. No statistically significant association was found between the placement of animals and size and gender. 3. Results The chi-square test carried out on the individuals of A. vulgare in- dependently of their aggregation state yielded no statistically sig- Table 1 summarizes the number and percentage of the animals nificant relationship between micro-vibrations and the sector where within each tested group, overall and stratified by gender, size, vibra- animals locate themselves (df = 1, chi-square = 1.00, p = 0.32, tional intensity sector, and aggregation state. Cohen’s w = 0.30). The aggregation dynamics after 5, 30, 60 and 120 min from the The possible associations between the placement of the individuals release of the animals are shown per group in Figs. 2 and 3. The non- of A. vulgare inside the test apparatus and the substrate-borne vibrations exposed individuals of A. officinalis already tended to aggregate in a were then researched more thoroughly with ordinal logistic regression unique point of the sector H, in the first half an hour from the release models with and without interactions. All interactions were statistically (Fig. 2a). The aggregate progressively increased and consolidated after non-significant, and the corresponding models were not considered for an hour, for becoming stable completely after two hours, leaving very a parsimony issue. As earlier, the models without interactions had very few animals outside. The exposed group of A. officinalis (Fig. 2b) similar values of the fit statistics for the comparison of nested models showed from the first minutes a greater dispersion compared tothe (QIC). Thus, we chose the full model as biologically more informative. control group inside the arena. After 30 min, one could notice an at- Table 3 reports the results of the full model. Our model yielded no tempt of aggregation, but in a confused enough way, which concluded statistically significant associations between the placement of animals with the formation of small aggregates and a higher number of isolated

425 S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430

Fig. 3. Outline of the aggregation dynamics of Armadillidium vulgare (AV) not exposed (a) and exposed (b) to micro-vibrations, after 5, 30, 60 and 120 min from the release of the animals in the center of the arena. The source of micro-vibrations is indicated in black (■) or white (□) when it is OFF or ON, respectively.

Table 1 Table 2 Number and percentage of the animals within each tested group, overall and Results of the ordinal logistic regression model for predictors of the placement stratified by gender, size, vibrational sector, and aggregation state (in- of the individuals of A. officinalis inside a shared space. The outcome variable is dependently of the positioning sector). Group 1, smaller size; Group 2, larger ordinal with three levels of vibrational intensity (low sector = 0, medium size. H, high vibrational intensity; M, medium vibrational intensity; L, low vi- sector = 1, high sector = 2). The parameter estimates were calculated with the brational intensity. generalized estimating equations (GEEs) methodology to take into account the correlation among observations. Group 1, smaller size; Group 2, larger size. Non-exposed Exposed Non- Exposed SE = standard error, OR = odds ratio, CI = confidence interval. A. officinalis A. officinalis exposed A. vulgare N (%) N (%) A. N (%) Predictor SE() Z (p) OR (95% CI) vulgare N (%) State Exposed A. −3.97 0.59 −6.69 0.02 Males 28 (38.4) 38 (52.1) 36 (49.3) 65 (89.0) officinalis (< .0001) (0.01–0.06) Group 1 14 (19.2) 22 (30.1) 19 (26.0) 31 (42.5) Non-exposed A. Ref. Sector H 14 (19.2) 7 (9.6) 9 (12.3) 17 (23.3) officinalis Sector M 0 (0.0) 9 (12.3) 6 (8.2) 4 (5.5) Size Sector L 0 (0.0) 6 (8.2) 4 (5.5) 10 (13.7) Group 1 0.74 0.44 1.68 2.09 Group 2 14 (19.2) 16 (21.9) 17 (23.3) 34 (46.6) (0.09) (0.88–4.93) Sector H 12 (16.4) 5 (6.8) 9 (12.3) 10 (13.7) Group 2 Ref. Sector M 2 (2.7) 1 (1.4) 2 (2.7) 10 (13.7) Gender Sector L 0 (0.0) 10 (13.7) 6 (8.2) 14 (19.2) Males −0.26 0.41 −0.64 0.77 Females 45 (61.6) 35 (47.9) 37 (50.7) 8 (11.0) (0.52) (0.35-1.71) Group 1 16 (21.9) 21 (28.8) 18 (24.7) 5 (6.8) Females Ref. Sector H 15 (20.5) 6 (8.2) 10 (13.7) 1 (1.4) Sector M 1 (1.4) 10 (13.7) 2 (2.7) 1 (1.4) Sector L 0 (0.0) 5 (6.8) 6 (8.2) 3 (4.1) Group 2 29 (39.7) 14 (19.2) 19 (26.0) 3 (4.1) Sector H 28 (38.4) 4 (5.5) 7 (9.6) 1 (1.4) square = 50.05, p < .0001). The pairwise two-sided multiple comparison Sector M 1 (1.4) 4 (5.5) 7 (9.6) 0 (0.0) analysis was carried out using the Dwass, Steel, Critchlow-Fligner (DSCF) Sector L 0 (0.0) 6 (8.2) 5 (6.8) 2 (2.7) method (Dwass, 1960; Steel, 1960; Critchlow and Fligner, 1991). We found All animals 73 (100) 73 (100) 73 (100) 73 (100) statistically significant differences between the positioning distances forthe Aggregated 68 (93.2)a 56 (76.7)b 58 (79.5)c 62 (84.9)d following comparisons: i) non-exposed A. officinalis and exposed A. offici- Isolated 5 (6.8) 17 (23.3) 15 (20.5) 11 (15.1) nalis (Wilcoxon mean score: 92.9 vs. 190.8, DSCF Value = 11.86,

a p < .0001), ii) A. officinalis and A. vulgare both non-exposed (Wilcoxon 1 aggregate in sector H (68 animals). b 1 aggregate in sector H (12 animals), 2 aggregates in sector M (24 animals), mean score: 92.9 vs. 153.7, DSCF Value = 6.37, p < .0001), and iii) ex- 2 aggregates in sector L (20 animals). posed A. officinalis and non-exposed A. vulgare (Wilcoxon mean score: 190.8 c 3 aggregates in sector H (31 animals), 1 aggregate in sector M (11 animals), vs. 153.7, DSCF Value = 3.81, p = 0.04) (Fig. 4). 2 aggregates in sector L (16 animals). Our results showed that individuals of A. officinalis seem to react d 2 aggregates in sector H (27 animals), 1 aggregate in sector M (8 animals), significantly to the presence of micro-vibrations, positioning themselves 2 aggregates in sector L (27 animals). preferentially far away from the vibrational source. In contrast, in- dividuals of A. vulgare seem to show a similar behavior at the level of inside the test apparatus and micro-vibrations, gender, and size. placement inside the test apparatus in both absence and presence of We evaluated any possible differences at the level of positioning dis- micro-vibrations. tances (in cm) of the animals from the source of micro-vibrations in the four The relationships between the number of aggregates or isolated tested groups with the Kruskal-Wallis nonparametric test (df = 3, chi- subjects and the exposure to micro-vibrations were studied with

426 S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430

Table 3 vibrations is about 6-times the number of aggregates in the absence of Results of the ordinal logistic regression model for predictors of the placement micro-vibrations (model 1). The expected number of isolated subjects in of the individuals of A. vulgare inside a shared space. The outcome variable is the presence of micro-vibrations is nearly 12-times the number of iso- ordinal with three levels of vibrational intensity (low sector = 0, medium lated subjects in the absence of micro-vibrations (model 2). Further- sector = 1, high sector = 2). The parameter estimates were calculated with the more, model 1 also found a statistically significant association between generalized estimating equations (GEEs) methodology to take into account the the number of aggregates and the number of isolated subjects, holding correlation among observations. Group 1, smaller size; Group 2, larger size. the vibration variable constant. A one-unit increase in the number of SE = standard error, OR = odds ratio, CI = confidence interval. isolated subjects yields a 5% decrease in the estimated mean of ag- Predictor SE() Z (p) OR (95% CI) gregates number. For A. vulgare, models 3 and 4 highlighted no statis- tically significant association between both the number of aggregates State and of isolated subjects and micro-vibrations. Model 4 found a statis- Exposed A. −0.54 0.35 −1.52 0.59 vulgare (0.13) (0.29–1.17) tically significant association between the number of isolated subjects Non-exposed Ref. and both number of aggregates and number of aggregated subjects, A. vulgare holding all other variables constant. A one-unit increase in the number Size of aggregates yields a 58% increase in the estimated mean of isolated Group 1 0.40 0.31 1.30 1.50 (0.19) (0.81–2.76) subjects number. A one-unit increase in the number of aggregated Group 2 Ref. subjects yields a 7.4% decrease in the estimated mean of isolated sub- Gender jects number. Males 0.34 0.38 0.91 1.41 Our results seem to indicate that, unlike A. vulgare, both number of (0.36) (0.67–2.96) aggregates and number of isolated subjects significantly increase for A. Females Ref. officinalis when exposed to micro-vibrations compared to when non- exposed. For A. officinalis, furthermore, the expected number of ag- gregates slightly but significantly decreases with the increase in isolated subjects. For A. vulgare, the number of isolated subjects increases with Poisson regression with robust standard errors and with generalized the increase in aggregates number and decreases with the increase in distribution. The choice of the models with the best fitting was mainly aggregated subjects, significantly in both cases. based on the fit statistics (QIC), or on the likelihood ratio (LR) testfor nested models. Table 4 shows the results of our best-fitting models. All 4. Discussion models are adjusted except model 3. For A. officinalis, models 1 and 2 yielded a statistically significant association between both the number This exploratory study pointed out a statistically significant asso- of aggregates and of isolated subjects and micro-vibrations. In parti- ciation between the aggregative behavior of individuals of A. officinalis cular, the expected number of aggregates in the presence of micro- sharing a small space and the presence of substrate-borne vibrations. No

Fig. 4. Distribution of Wilcoxon scores for the distance (in cm) from the source of micro-vibrations in the four tested groups of animals. Kruskal-Wallis test and pairwise two-sided multiple comparisons with the Dwass, Steel, Critchlow-Fligner (DSCF) method. Abbreviations: AO, Armadillo officinalis; AV, Armadillidium vulgare.

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Table 4 preferential defense strategy. Secondly, the presence of substrate-borne Results of the Poisson models with robust standard errors and the generalized vibrations might interfere somehow with a possible intraspecific com- Poisson model for predictors of the number of aggregates and isolated subjects munication of vibrational type, meant as hypothetical signaling among in A. officinalis and A. vulgare. Models 1, 2 and 4 are adjusted; model 3 is un- conspecifics, able to lead to the formation of large dimension ag- adjusted. SE = standard error, CLs = confidence limits. gregates. Indeed, in the absence of micro-vibrations, A. officinalis seems A. officinalis to show a better capability of aggregation compared to A. vulgare forming a unique, large aggregate including most of the subjects pre- Model 1: Poisson regression with robust SEs - Outcome: n. of aggregates per sent inside the arena, as if this species has a better capability of con- sector specifics recognition. Besides a possible chemical signal of recognition Parameter SE() 95% CLs Z p-value such as an aggregation pheromone (Broly et al., 2012), a form of like- stridulation mechanical signaling produced during the friction of the Intercept −1.01 0.81 −2.60 0.57 −1.25 0.2107 bodies inside the aggregate might be involved too. Elias and Mason Vibrations 1.80 0.80 0.23 3.37 2.25 0.0248 (2010) reported that the substrate-borne vibrations produced by stri- No Vibrations 0.00 (Ref.) dulation have a higher frequency compared to those produced by tre- N. of isolated −0.05 0.02 −0.10 −0.01 −2.19 0.0287 subjects mulation. Hence, the friction of the bodies of the animals inside an aggregate might produce substrate-borne vibrations easily identifiable Model 2: Generalized Poisson regression - Outcome: n. of isolated subjects per from isolated individuals and provide, in this way, an additional ag- sector gregation signal besides a possible chemical stimulus. Sensory receptors

Parameter 95% CLs Z p-value might be involved. In the legs of Apis mellifera carnica (Hymenoptera, Apidae), Sandeman et al. (1996) found particular receptors able to Intercept 0.88 0.67 −0.43 2.19 1.32 0.1877 detect micro-vibrations propagating on the combs of its nests. No spe- Vibrations 2.47 1.16 0.19 4.75 2.13 0.0335 cific receptor with these characteristics has, however, been sofarde- No Vibrations 0.00 (Ref.) scribed in terrestrial isopods, unlike other invertebrates (Barth, 1982; N. of aggregates −1.08 0.71 −2.48 0.33 −1.51 0.1323 Hutchings and Lewis, 1983; Kalmring, 1985; Popper et al., 2001; Scale Parameter 0.49 0.46 Devetak et al., 2004). A. vulgare It is our opinion that A. officinalis being a species able to emit stri- dulations might also be able to manage substrate-borne vibrations for Model 3: Poisson regression with robust SEs - Outcome: n. of aggregates per multiple goals and in a better way than a non-stridulating species as A. sector vulgare. Hence, A. officinalis might be advantaged both as a defense Parameter 95% CLs Z p-value strategy from predators and as a capability of interpreting substrate- borne vibrations inside a wider and more complex signaling network of Intercept 0.69 0.24 0.23 1.16 2.94 0.0033 intra- and interspecific communications, like in insects. This higher Vibrations −0.18 0.29 −0.74 0.38 −0.64 0.5249 sensitivity to micro-vibrations might be a more sophisticated form of No Vibrations 0.00 (Ref.) evolutionary adaptation to the xeric environment, being the latter Model 4: Poisson regression with robust SEs - Outcome: n. of isolated subjects particularly unfavorable to these animals at the level of water retention per sector and predation. Both terrestrial isopods and insects (e.g., cockroaches) emerged in Parameter 95% CLs Z p-value the Late Paleozoic (Broly et al., 2013) evolving in parallel likely from an aquatic pancrustacean ancestor (Regier et al., 2005, 2010) so that these Intercept 2.13 0.14 1.87 2.40 15.66 < .0001 taxa might share similar physiological and behavioral mechanisms for Vibrations −0.13 0.09 −0.31 0.05 −1.39 0.1646 No Vibrations 0.00 (Ref.) both aggregation and communication processes. The presence of a N. of aggregates 0.46 0.23 0.01 0.91 2.01 0.0443 stridulatory apparatus could be considered a phenomenon of con- N. of aggregated −0.08 0.02 −0.11 −0.04 −4.41 < .0001 vergent evolution, for instance, with insects. Anyway, as previously subjects stated, this stridulatory apparatus is present in all the species of the genus Armadillo and at least in another species of the genus Cubaris. Hence, we can hypothesize one common ancestor within the family Armadillidae bearing a similar stridulatory apparatus. Gregarious behavior and aggregative phenomena in woodlice are statistically significant association was instead found for A. vulgare, the thought to be mainly related to a mechanism aimed to prevent de- species without stridulatory organ. siccation (Allee, 1926; Brockett and Hassall, 2005; Broly et al., 2014), Our results seem to indicate that individuals of A. officinalis but other possible implications have also been proposed (Broly et al., significantly react to the presence of the vibrational impulse, showing 2013). a higher sensitivity than individuals of A. vulgare and prevalently Aggregation might be a shared form of defense from predators that positioning themselves in the zones with the lowest vibrational in- favors secretion-based repulsive strategies (Schmalfuss, 1984). Fur- tensities as if micro-vibrations are perceived as a source of danger and/ thermore, being most of the preyed terrestrial isopods in the juvenile or disturbance. Furthermore, unlike A. vulgare, both number of ag- stage (Sunderland and Sutton, 1980), the massive and synchronized gregates and of isolated subjects significantly increase in the presence release of juveniles from females belonging to an aggregate might be of substrate-borne vibrations than in the absence of micro-vibrations, another form of defense strategy and survival (Ims, 1990; Broly et al., which would seem to indicate that animals appear a bit disoriented in 2013). For A. officinalis, we found that juveniles exposed to micro-vi- their aggregative capability. brations carry out, on average, a significantly lower number of turn For A. officinalis, these results may thus lead to the following two alternations, meant as a hypothetical mechanism of defense against speculations. Firstly, the observed behavior might be related to the adverse conditions, than exposed and non-exposed adults and they are perception of substrate-borne vibrations as a coming danger, leading to also less fast (Cividini and Montesanto, 2018b). These results might in higher dispersion and escape from the source of disturbance as a part explain their greater vulnerability towards predators, and the need

428 S. Cividini, G. Montesanto Behavioural Processes 157 (2018) 422–430 for protection by the group. Acknowledgments Sheltering behavior is another form of aggregation favoring copro- phagy as a secondary food source for growth and survival (Hassall and Special thanks to Prof. Olga Lazareva (editor-in-chief) and the Rushton, 1982; Hassall et al., 2005). Juveniles, eating the feces of anonymous reviewers who checked over our manuscript for their va- adults, are colonized by symbiotic hepatopancreatic bacteria (Zimmer luable suggestions. and Topp, 1998; Wang et al., 2007). Other possible implications justi- fying aggregation might be related to facilitating of the meeting be- Appendix A tween males and receptive females for reproduction and to a reduction of oxygen consumption (Allee, 1926; Takeda, 1984) although the latter Settings to generate the micro-vibrations used in the experiment with the seems more to be interpreted as a consequence of aggregation. software Audacity (ver. 1.2.4) We think that this study has contributed to clarifying some inter- esting, novel aspects of still little known behavioral processes in ter- The option Noise was selected starting from the menu Generate. restrial isopods, pointing out the possibility of the existence of an as- In the window named Noise Generator, the following parameters sociation between the aggregative behavior of A. officinalis and the were set: exposure to substrate-borne vibrations. No statistically significant evi- dence was found for A. vulgare, a species not able to emit stridulations, • Noise type: Brownian unlike A. officinalis. The high reactivity to micro-vibrations of A. offi- • Amplitude: 1 cinalis compared to other species might fit into a more complex and • Duration: 3 minutes wider network of communications, both intraspecific, e.g., as a signal of aggregation, and interspecific, e.g., as a more sophisticated defense The sound was normalized at −4.0 dB with the command strategy from predators. Communication mediated by substrate-borne Normalize, in the Effect menu. vibrations in insects, for instance, has acquired more and more im- portance compared to what was previously thought (Virant-Doberlet References and Čokl, 2004). Indeed, this kind of communication seems to be in- volved in many adaptive roles in both invertebrates and vertebrates Agodi, A., Oliveri Conti, G., Barchitta, M., Quattrocchi, A., Lombardo, B.M., Montesanto, (Hill, 2001, 2008, 2009; Hill and Wessel, 2016) such as mating, par- G., Messina, G., Fiore, M., Ferrante, M., 2015. Validation of Armadillo officinalis Dumèril, 1816 (Crustacea, Isopoda, Oniscidea) as a bioindicator: in vivo study of air ental cares, foraging, competition, and danger perception. benzene exposure. Ecotox. Environ. Safe. 114, 171–178. Terrestrial isopods show different habitus types for their defensive Allee, W.C., 1926. Studies in animal aggregations: causes and effects of bunching in land strategies, namely, runner-type, clinger-type, creeper-type, spiny-type isopods. J. Exp. Zool. 45, 255–277. Barth, F.G., 1982. Spiders and vibratory signals: sensory reception and behavioral sig- and roller-type. 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