Abstract Autotomy, the voluntary shedding or detachment of a body part at a determined cleavage plane, is a common anti-predation defense mechanism in several animal taxa, including arthropods. Among arachnids, autotomy has been observed in harvestmen, mites, and spiders, always involving the loss of legs. Autotomy of the opisthosoma (abdomen) was recently reported in a single species of the Neotropical buthid scorpion genus Ananteris Thorell, 1891, but few details were revealed. Based on observations in the field and laboratory, examination of material in museum collections, and scanning electron microscopy, we document autotomy of the metasoma (the hind part of the opisthosoma, or ‘tail’) in fourteen species of Ananteris. Autotomy is more common in males than females, and has not been observed in juveniles. When the scorpion is held by the metasoma, it is voluntarily severed at the joints between metasomal segments I and II, II and III, or III and IV, allowing the scorpion to escape. After detachment, the severed metasoma moves (twitches) automatically, much like the severed tail of a lizard or the severed leg of a spider, and reacts to contact, even attempting to sting. The severed surface heals rapidly, scar tissue forming in five days. The lost metasomal segments and telson cannot be regenerated. Autotomy of the metasoma and telson results in permanent loss of the posterior part of the scorpion’s digestive system (the anus is situated posteriorly on metasomal segment V) and the ability to inject venom by stinging. After autotomy, scorpions do not defecate and can only capture small prey items. However, males can survive and mate successfully for up to eight months in the laboratory. In spite of diminished predation ability after autotomy, survival allows males to reproduce. Autotomy in Ananteris therefore appears to be an effective, adaptive, anti-predation escape mechanism.

Citation: Mattoni CI, García-Hernández S, Botero-Trujillo R, Ochoa JA, Ojanguren-Affilastro AA, Pinto-da-Rocha R, et al. (2015) Scorpion Sheds ‘Tail’ to Escape: Consequences and Implications of Autotomy in Scorpions (Buthidae: Ananteris). PLoS ONE 10(1): e0116639. https://doi.org/10.1371/journal.pone.0116639 Academic Editor: Jordi Moya-Larano, Estacion Experimental de Zonas Áridas (CSIC), SPAIN Received: May 26, 2014; Accepted: December 11, 2014; Published: January 28, 2015 Copyright: © 2015 Mattoni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Financial support was provided by: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, http://www.fapesp.br), grant numbers 2008/06604-7 and 2012/02969-6 to RPR; and Agencia Nacional de Promoción Científica y Tecnológica (http://www.agencia.mincyt.gob.ar), grant numbers PICT 2010-1764 to AAOA, PICT 2006-750 and 2010-0906 to CIM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Autotomy is the process by which some animals voluntarily shed or detach a body part, usually as an anti-predator defense mechanism. In order to be considered autotomy, the process of shedding or detachment must be provoked by external stimuli, achieved by an intrinsic mechanism, and mediated by the nervous system [1–3]. Autotomy occurs along permanent sites of weakness, cleavage planes that permit a clean break when a body part is detached, with anatomical features that minimize trauma and promote rapid sealing of the fluid compartment, leading to swift closure and healing of the wound [2, 4]. Among invertebrates, autotomy is characterized by limited loss of hemolymph from either the stump or discarded appendage [3]. The separation of an appendage from the body at a site of weakness, when pulled by an outside agent, has been termed autospasy by some authors (e.g. [5, 6]), and is here considered synonymous with autotomy. The detached body part, e.g., the tail of lizards or the legs of arthropods [7, 8], may also act as a distraction, engaging the predator’s attention by spontaneously twitching, writhing or wriggling, while the animal escapes. The incidence of autotomy in natural populations may be determined by predation efficiency and intensity, anatomical mechanisms, microhabitat preference, sex and ontogenetic differences, intraspecific aggression and the use of other defense mechanisms [3, 8]. Autotomy of a body part is an effective anti-predator defense mechanism that evolved independently in various taxa [3]. The phenomenon has been recorded in cnidarians, annelids, molluscs, echinoderms, arthropods and vertebrates [3, 7, 9]. Among arthropods, autotomy has been reported in crustaceans, hexapods, chilopods and arachnids, always involving the loss of appendages, usually legs [10–14]. Among arachnids, autotomy of the legs has been observed in Opiliones (harvestmen), Acari (mites and ticks), and Araneae (spiders) [3, 5, 6, 15], but was not reported to occur in scorpions. Autotomy of the metasoma, the posterior part of the opisthosoma, or ‘tail’, was recently reported in several species of the Neotropical buthid scorpion genus Ananteris Thorell, 1891 [16–18], which currently comprises 79 species of small (9 to 42 mm in total adult length), cryptic, terrestrial scorpions inhabiting the tropical forests of northern South America, from Costa Rica to Argentina [19–26]. These reports represent the first cases of autotomy of opisthosoma (abdomen) in arthropods. Based on observations in the field and laboratory, examination of material in museum collections, and scanning electron microscopy, we document autotomy of the metasoma in fourteen species of the genus, and provide behavioral observations with the following objectives: (1) to verify that metasomal autotomy in Ananteris is provoked by external stimuli, achieved by an intrinsic mechanism, and mediated by the nervous system; (2) to confirm that cleavage planes are present in the metasoma of Ananteris; (3) to assess whether the incidence of autotomy differs between the sexes; and (4) to provide data about post-autotomy behavior which may illuminate the functional significance of metasomal autotomy.

Materials and Methods Collecting permits Collecting permits were issued by the following agencies: Brazil, Ministério do Meio Ambiente, Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio #17974-3), and Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA #10148-1); Bolivia, Ministerio de Medio Ambiente y Agua (MMAyA VMA-DGBAP #1319); Perú, Direccion General de Forestal y Fauna Silvestre (#002-2008-INRENA-IFFS-DCB); Ecuador, Ministerio de Ambiente (scientific research authorization #007-14 IC-FAU-DNB/MA); Argentina, Secretaría de Ambiente de la Provincia de Córdoba (#SECA01-524433053-908). Specimens from Colombia belong to registered biological collections and did not require of specific permissions for this study. Specimens from Venezuela are part of old collections for which no permissions could be traced. This study did not involve any endangered or protected species. Field observations of autotomy Ananteris specimens were collected in Argentina, Bolivia, Brazil, Colombia, Peru and Venezuela (details of collection localities in S1 Appendix) by turning stones during the day, or by ultraviolet (UV) light detection at night [27], using portable UV lamps, comprising mercury vapor tubes attached to a chromium reflector, and powered by a 12V, 7 Amp/hour battery, or Maglite flashlights modified with UV LED attachments. Autotomy was recorded when part of the metasoma was shed by an individual during collection. When autotomy occurred, the sex, age (adult or juvenile), and site of detachment (metasomal segments on either side of the cleavage plane, Fig. 1A), were noted. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Autotomy in Ananteris Thorell, 1891 scorpions. A. Ananteris balzani Thorell, 1891, adult male from Serra das Araras Ecological Station, Mato Grosso State, Brazil. Dashed lines indicate autotomy cleavage planes between metasomal segments I-IV. B, C. Autotomy in Ananteris solimariae Botero-Trujillo & Flórez, 2011, adult male, video frames. B. Exact moment before autotomy, scorpion fighting to escape. Arrow indicates beginning of cleavage. C. Immediately after autotomy, detached tail twitching. https://doi.org/10.1371/journal.pone.0116639.g001 Museum records of autotomy The scorpion collections of several museums were searched for Ananteris specimens that had undergone autotomy prior to collection (complete list of material examined in S1 Appendix). Autotomy was recorded as present when part of the metasoma (including telson) was missing, and a brown scar was evident at its severed stump (as Ananteris specimens become brittle after ethanol fixation, we could not be confident that autotomy had occurred if the metasoma was broken but no scar was evident). The species, sex, age, site of detachment, and presence of a scar, were noted for each specimen. The incidence of autotomy, i.e., the percentage of scorpions with part of the metasoma (including telson) missing and a scar present at the severed stump, in a single population was calculated for those species with more than ten specimens from a single collection locality. The results for different populations of the same species were not combined because it was assumed that the incidence of autotomy could vary among localities due to ecological factors (e.g., presence and abundance of predators). Differences among sexes and stages (adult males vs adult females vs immatures) were analyzed as 2×3 contingency tables with Fisher’s exact test in R v. 3.1.1 statistical package [28]. Experimentally-induced autotomy Twenty-five adult males and five adult females of Ananteris solimariae Botero-Trujillo & Flórez, 2011 were collected for experimentation from a population at Girón, Santander Department, Colombia. One of the females, gravid when captured, gave birth, and two second instar juveniles from her litter were also used in the experiments. Scorpions were housed separately in plastic containers (7 cm diameter; 10 cm height) with moistened cotton as water supply and fed every two weeks with crickets, Gryllodes sigillatus Walker, 1869. After 7 to 10 days of acclimation, each adult scorpion was placed separately in a plastic container with a rough surface (humid cotton), and subjected to the following treatment, designed to demonstrate if detachment was provoked by external stimuli and achieved by an intrinsic mechanism. The metasoma was held with forceps on segments III, IV or V and gently pulled backwards to simulate capture by a predator, for no more than 30 seconds (sufficiently short duration to categorize the phenomenon as autotomy). The time to detachment of the metasomal segments was noted, if applicable. In order to avoid damaging the small second instar juveniles with the forceps, the posterior part of the metasoma was instead attached to sticky tape, which was pulled backwards with the forceps. Ten adult males were subjected to the same treatment, but using glass Petri dishes as a substrate, and the same specimens were held in the air for 30 seconds, without being allowed to contact any substrate. Experiments were filmed with a SONY Cyber-shot DSC-W35 camera and photographs taken with a Canon EOS Rebel T2i camera fitted with a 50 mm macro lens. The healing process of the wound was also documented, by photographing the development of scars on the severed stump of the metasoma during successive time intervals. The effect of anesthesia on autotomy was also investigated, to confirm whether autotomy is mediated by the nervous system, by placing another five adult male individuals of A. solimariae in a styrofoam box with ice, prior to manipulation of the metasoma. Differences between the experiments (adult males on rough substrate vs adult females on rough substrate; adult males on rough substrate vs adult males in Petri dishes; adult males on rough substrate vs adult males held in the air; adult males on rough substrate vs anesthetized adult males on rough substrate) were analyzed as 2×2 contingency tables with Fisher’s exact test in R v. 3.1.1 [28]. Additional experiments, identical to those performed on A. solimariae, were conducted with 92 live individuals (32 adult males, 32 adult females, 28 immatures) of ten other scorpion species in seven genera and three families: Bothriuridae Simon, 1880: Bothriurus cordubensis Acosta, 1995: 3 adult males, 2 adult females; Bothriurus flavidus Kraepelin, 1911: 2 adult males, 1 adult female, 3 immatures; Brachistosternus ferrugineus Thorell, 1876: 4 adult males, 5 adult females, 5 immatures; Timogenes elegans Mello-Leitão, 1931: 4 adult males, 1 adult female; Timogenes dorbignyi Guérin Méneville, 1843: 3 adult males, 1 adult female; Urophonius brachycentrus Thorell, 1876: 2 adult males, 4 adult females, 3 immatures; Buthidae C.L. Koch, 1837: Tityus trivittatus Kraepelin, 1898: 5 adult females; Zabius fuscus Thorell, 1876: 8 adult males, 6 adult females, 4 immatures; Zabius birabeni Mello-Leitão 1938: 4 adult males, 2 adult females; Hormuridae Laurie, 1896: Opisthacanthus elatus Gervais, 1844: 2 adult males, 5 adult females, 13 immatures. The bothriurid and buthid specimens were collected at several localities in Córdoba Province, Argentina, whereas the O. elatus specimens were collected in Santander Department, Colombia, in 2012. SEM of cleavage plane and scar In order to determine whether a defined cleavage plane exists in the metasoma, six ethanol-preserved specimens of three buthid species (two adult specimens per species), Ananteris balzani Thorell, 1891, Tityus uruguayensis Borelli, 1901 and Z. fuscus, the last two species included for comparison with A. balzani, were manipulated with forceps to induce detachment of the metasoma between segments II and III, and III and IV. Scanning electron micrographs (SEM) were taken of the sites of detachment in these specimens, as well as of the scarred, post-autotomy metasomal segments of two A. solimariae specimens, with a Philips XL30 TMP SEM. Samples for SEM were dehydrated and coated with gold-palladium in a Thermo VG Scientific SC 7620 sputter coater. Post-autotomy behavior The behavior of A. solimariae specimens post-autotomy was recorded in the laboratory and, when possible, compared with the behavior of intact (i.e., pre-autotomy) specimens. In order to assess the effect of losing part of the metasoma on male mating success, four mating trials were conducted with two adult females and four adult males, two with the metasoma intact and two without the last three metasomal segments. Each pair was placed in a mating arena (20 × 40 × 30 cm) with a substrate comprising soil, stones and pieces of tree bark from the collection locality. Mating behavior was observed and filmed under a 40 W red lamp. Two mating trials were conducted per female. The first two trials, involving males with an intact metasoma, were conducted when the females were gravid. The second and third trials, involving post-autotomy males, were conducted several months later, after both females had given birth and the juveniles had left their mothers.

Discussion Metasomal detachment in Ananteris meets the criteria for defensive autotomy [2, 3]. Detachment is provoked by external stimuli and achieved by an intrinsic mechanism: in order to occur, the metasoma must be grasped by a potential predator (simulated by holding it with forceps in the experiments presented here) and the scorpion must pull forward with its pedipalps and/or legs contacting a rough surface, allowing the metasomal segments to separate along permanent sites of weakness (cleavage planes). The lateral twisting motion of the metasomal segments anterior to the cleavage plane, and the fact that autotomy did not occur in anesthetized specimens, suggest that the process is mediated and controlled by the nervous system. Autotomy in Ananteris provides a mechanism for escape from predation. Movements of the detached metasomal segments are presumably controlled by the last or second-last of the four neural ganglia, situated anteriorly in each of the first four metasomal segments. The fourth ganglion has a pair of nerves, extending posteriorly into two branches, one innervating metasomal segment V and the other innervating the telson [30], which provide these detached segments with the ability to react to stimuli, increasing the effectiveness of distraction. There are several costs associated with autotomy in Ananteris. A potentially significant cost, post-autotomy, is the loss of part of the digestive system, including the posterior part of the mesenteron (midgut or middle intestine), the entire proctodeum (hindgut or posterior intestine, contained in metasomal segment V), and the anus, which opens at the posteroventral end of segment V, in the intersegmental membrane preceding the telson [30]. The only visible effect of losing part of the digestive system appears to be the accumulation of excrement inside the mesosoma, caused by the inability to defecate. This is the first report, to our knowledge, of a case in which autotomy prevents defecation. The ability of scorpions to excrete very little waste, consisting mostly of insoluble nitrogenous compounds [31], may permit their survival despite this handicap. Furthermore, it may be possible to spontaneously release accumulated excrement during at least one additional autotomy event, as described above. The observed cases of a second autotomy, taken together with the finding that most cases of autotomy occurred between metasomal segments III and IV, suggest that autotomy of more posterior metasomal segments (i.e., between segments III and IV rather than II and III or I and II) is selectively advantageous for several reasons. Detachment of the metasoma between segments III and IV appears to leave more intestinal space for accumulation of excrement, and may provide a chance to release accumulated excrement during a second or third autotomy event, as well as an additional chance of escape from predators. Individuals that undergo autotomy between segments III and IV may survive longer, allowing more opportunity to increase their reproductive success, than those which undergo autotomy between segments II and III or I and II, in part because of the potential for additional autotomy events. Loss of the telson, which bears the venom gland and aculeus (sting), negatively affects the scorpion’s ability to catch larger prey or to sting potential predators, and considerably reduces its defense capabilities, but post-autotomy scorpions can still capture and feed successfully on smaller prey. A similar observation has been reported in crabs with autotomy of the chelipedes [32]. Another possible cost of autotomy could be the loss of metasomal photoreceptors, which have been identified in a few scorpion species to date [33, 34]. These photoreceptors differ from the median and lateral ocelli in their sensitivity to wavelengths of light [35] and may assist with phototaxic behavior [36]. Their presence in Ananteris has not yet been determined, however. Although autotomy results in permanent loss of the posterior part of the digestive system and the ability to inject venom by stinging, among other possible costs, Ananteris scorpions are able to survive and mate successfully. A complete sequence of courtship behavior has not yet been described in any species of Ananteris, hence is impossible to know whether male Ananteris perform ‘clubbing’ (striking the partner with the metasoma while the sting is tucked away [29]) or the ‘sexual sting’ (male punctures the female’s body with his aculeus [29]). Nevertheless, two males with an incomplete metasoma successfully completed sperm transfer. As males are able to survive several months after autotomy, they have time to increase their reproductive success by mating with multiple females for as long as they remain alive. In the absence of life history data (including natural lifespan) for any species of Ananteris, it is impossible to know whether autotomy actually shortens the lifespan but even if that were the case, autotomy could potentially increase male survival and reproductive success, by allowing males to escape from predators and mate on more occasions. Autotomy may be adaptive because it allows Ananteris scorpions to survive predation. Although we have data for few species and populations, the incidence of autotomy in the field was low (up to 8.33%, increasing to 14.29% when only adult males are considered) and restricted to adult males. The higher incidence of autotomy in adult males may be explained by the difference in breeding behavior between the sexes. Male scorpions are more vagile, wandering in search of females during the breeding season, placing them at greater risk of predation [15]. The difference in the incidence of autotomy between the sexes may also be attributed to differences in survival and reproductive success. In most scorpion species, adult males live no more than one or two reproductive seasons, whereas adult females live much longer [15]. Autotomy may reduce the space available for a female’s embryos (and hence the size of her litter) due to an accumulation of excrement in the opisthosoma, resulting in a decrease in reproductive success, compared with escaping from a predator intact. Furthermore, females could be less predisposed to undergo autotomy to avoid losing the sting, which greatly enhances predation ability. Females need more food for embryonic development during gestation. Including Ananteris mauryi Lourenço, 1982 [18], at least fifteen species of Ananteris exhibit autotomy, which may prove to be synapomorphic for the genus, but further observations on the remaining species and/or a phylogenetic hypothesis confirming their monophyly are needed to confirm this hypothesis. Research into the costs and benefits of metasomal autotomy in Ananteris are also needed, to explore how trade-offs may have influenced its evolution, and the occurrence of autotomy should be investigated in other scorpion taxa.

Acknowledgments We thank Eduardo Flórez (Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá, Colombia), Giovanny Fagua and Igor Dimitri Forero (Museo Javeriano de Historia Natural “Lorenzo Uribe S.J.”, Pontificia Universidad Javeriana, Bogotá, Colombia), and Claudia Medina (Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Colombia) for lending material from or otherwise assisting with the study of material at their institutions; John Uribe for assisting with the production of photos and videos; Glauco Machado (Universidade de São Paulo, Brazil) for permitting use of the scorpion collection in his care; Oscar Javier Cadena Castañeda (Universidad Distrital Francisco José de Caldas, Bógota, Colombia) for identification of crickets; Alvaro Barragán and Emilia Moreno (Pontificia Universidad Católica del Ecuador) for facilitating research in Ecuador; the relevant permitting authorities in Argentina, Bolivia, Brazil, Ecuador and Perú for permission to collect scorpions in their respective countries; and Jordi Moya-Laraño and two anonymous reviewers for comments that improved the manuscript.

Author Contributions Conceived and designed the experiments: CIM SGH. Performed the experiments: CIM SGH. Analyzed the data: CIM SGH RBT JAO AAOA RPR LP. Contributed reagents/materials/analysis tools: CIM SGH RBT JAO AAOA RPR LP. Wrote the paper: CIM SGH RBT JAO AAOA RPR LP.