RESEARCH ARTICLE Passive and Active Defense in Toads: The Parotoid Macroglands in Rhinella marina and Rhaebo guttatus PEDRO L. MAILHO‐FONTANA1, MARTA M. ANTONIAZZI1, LUÍS F. TOLEDO2, VANESSA K. VERDADE3, JULIANA M. SCIANI1, KATIA C. BARBARO1, DANIEL C. PIMENTA1, 4 1 MIGUEL T. RODRIGUES , AND CARLOS JARED * 1Instituto Butantan, São Paulo, Brazil 2Museu de Zoologia, Universidade Estadual de Campinas, Campinas, Brazil 3Centro de Ciências Naturais e Humanas, Universidade Federal ABC, Santo André, Brazil 4Departamento de Zoologia, Universidade de São Paulo, São Paulo, Brazil

ABSTRACT have many skin poison glands used in passive defense, in which the aggressor causes its own poisoning when biting prey. In some amphibians the skin glands accumulate in certain regions forming macroglands, such as the parotoids of toads. We have discovered that the toad Rhaebo guttatus is able to squirt jets of poison towards the aggressor, contradicting the typical defense. We studied the R. guttatus chemical defense, comparing it with Rhinella marina,a sympatric species showing typical toad passive defense. We found that only in R. guttatus the parotoid is adhered to the scapula and do not have a calcified dermal layer. In addition, in this species, the plugs obstructing the glandular ducts are more fragile when compared to R. marina.As a consequence, the manual pressure necessary to extract the poison from the parotoid is twice as high in R. marina when compared to that used in R. guttatus. Compared to R. marina, the poison of R. guttatus is less lethal, induces edema and provokes nociception four times more intense. We concluded that the ability of R. guttatus to voluntary squirt poison is directly related to its stereotyped defensive behavior, together with the peculiar morphological characteristics of its parotoids. Since R. guttatus poison is practically not lethal, it is possibly directed to predators' learning, causing disturbing effects such as pain and edema. The unique mechanism of defense of R. guttatus may mistakenly justify the popular myth that toads, in general, squirt poison into people's eyes. J. Exp. Zool. 9999A: XX–XX, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Mailho‐Fontana PL, Antoniazzi MM, Toledo LF, Verdade VK, Sciani JM, J. Exp. Zool. Barbaro KC, Pimenta DC, Rodrigues MT, Jared C. 2013. Passive and active defense in toads: The 9999A:1–13, 2013 parotoid macroglands in Rhinella marina and Rhaebo guttatus. J. Exp. Zool. 9999:1–13.

Grant sponsor: CAPES and CNPq‐INCTTox—Brazilian Federal Government. Correspondence to: Carlos Jared, Cell Biology Laboratory, Instituto The skin of amphibians is characterized by the presence of mucous Butantan, Av. Vital Brasil 1500, CEP 05503‐000 São Paulo, Brazil. glands, mainly associated with protection against desiccation, and E‐mail: [email protected] granular glands, or poison glands, associated with chemical Received 8 July 2013; Revised 6 September 2013; Accepted 13 defense against predators and microorganisms (Toledo and September 2013 Jared, '95; Hillman et al., 2009). DOI: 10.1002/jez.1838 Published online XX Month Year in Wiley Online Library In some species the poison glands are grouped into large and (wileyonlinelibrary.com). very conspicuous protuberances, such as the parotoid macroglands

© 2013 WILEY PERIODICALS, INC. 2 MAILHO‐FONTANA ET AL. of toads (Toledo and Jared, '95; Duellman and Trueb, '96; the interdisciplinary nature of this work, biochemical and Clarke, '97; Jared et al., 2009). These macroglands are located in pharmacological studies on the parotoid secretion of both species the post‐orbital region and, on the skin surface, can be identified by were also carried out. The poison of R. guttatus showed low the presence of large pores from which the poison is ejected toxicity when compared to that of R. marina. However, it causes (Hostetler and Cannon, '74; Toledo et al., '92; Toledo and Jared, '95; high levels of inflammation and pain. All data were analyzed in Almeida et al., 2007; Jared et al., 2009). The parotoids are light of the natural history and the differences in the defensive constituted by many alveoli formed by a resistant collagenous behavior of the two species. connective tissue. The alveolar disposition side by side gives the appearance of honeycomb to the whole assembly (Jared et al., 2009). Each alveolus bears a very large poison gland that MATERIALS AND METHODS communicates with the outside through a duct lined by a thick epithelial tissue forming a plug, which obstructs the duct and Six adult male R. marina (Fig. 1a) with snout‐vent length (SVL) avoids poison release, acting as a cork in a bottle (Jared (114.91 2.67 mm) and six adult Rhaebo guttatus (Fig. 1c) (SVL et al., 2009). 115.52 11.3mm) were respectively collected in Santarém and When toads are disturbed, they inflate the lungs and assume a Jacareacanga, state of Pará, Brazil. Male Swiss mice, weighing 18– stereotyped posture, offering the parotoids to the aggressor 20 g, were used in pharmacological experiments. All (Toledo and Jared, '95; Jared et al., 2009; Toledo et al., 2011). The procedures were performed in accordance with the standards of parotoids then, due to the internal pressure coming from the lungs, the Ethics Committee on Animal Use of Instituto Butantan become very turgid and ready to trigger poisonous shots in case (CEUAIB) (protocol #837/11). any external mechanical pressure is exerted, such as the bite of an aggressor (Jared et al., 2009). The abrupt pressure exerted by the Behavior bite is transferred to the alveoli leading to the rupture of the To obtain behavioral data, the animals were gently stimulated epithelial plugs and causing the expulsion of the poison in the in their natural environment, using a twig or a finger. This form of jets (Jared et al., 2009). Therefore, it is the predator who procedure followed the protocol of Toledo et al. (2011), applied causes its own poisoning, which can often be lethal (Sakate and in other behavioral studies and does not cause any harm to the Lucas de Oliveira, 2000; Jared and Antoniazzi, 2009; Jared toads. et al., 2009). Predator's self‐poisoning is characteristic of amphibian defense, which is classified as a “passive defense” Morphology (Jared and Antoniazzi, 2009; Antoniazzi et al., 2013), as opposed The animals were euthanized with a lethal dose of thiopental and to “active defense” presented, for example, by the snakes, who fixed in Bouin or 4% formaldehyde for histology and scanning attack the aggressor and inject their venom through glands electron microscopy (SEM), or in Karnowsky ('65), for transmis- compressed by surrounding muscles (Kochva, '78; Pough sion electron microscopy (TEM). et al., 2001; Jared et al., 2009). For anatomical examination of the fixed animals, the parotoids In Brazil, toads are represented by seven genera, Rhinella being were carefully detached from the dorsal skin for observation of the most representative. Rhinella marina is one of the most studied their relationship with the surrounding bones, muscles, and other toads, distributed from Texas, in North America, to the center of tissues. South America, in the Brazilian Amazon (Frost, 2013). Its defense For histology, fragments of dorsal skin and parotoids of both is typically passive, possessing well‐developed parotoids covering species were embedded in paraffin following routine histological almost the entire central part of the dorsal region. Another procedures. The scapular girdle of R. guttatus was also prepared common bufonid genus in the Amazonian region and Central following the same methods after decalcification in 4% ethyl- America is Rhaebo, from which the species R. guttatus is enediamine tetraacetic acid (EDTA) for 3 months. The sections of all widespread throughout the Brazilian Amazon. Recently it was samples (2‐ to 6‐mm thick) were stained with hematoxylin–eosin, reported that the defensive behavior of this species opposes to that Picrosirius or von Kossa (Bancroft and Steven, '90; Junqueira et al., found in other bufonids and other anurans in general. In contrast '79). The images were obtained by an Olympus BX51 microscope to the characteristic passive defense of amphibians, R. guttatus, and captured through a digital camera using the software Image‐ when disturbed, displays a series of stereotypical postures that Pro Express from MediaCybernetics (Rockville, MD, USA). surprisingly culminate with the voluntary ejection of its poison For SEM, fragments of the parotoids were dried in vacuum, towards the aggressor, at distances of up to 2 m (Jared et al., 2011). mounted in stubs and analyzed under low vacuum in a FEI Quanta The aim of this work was the comparative morphological study 250 microscope. For TEM, fragments of the parotoids were post‐ of the parotoids and their associated structures in the two toads in fixed in 1% osmium tetroxide, contrasted in 1% uranyl acetate order to understand the structural differences that lead to the and embedded in epoxy resin. Ultrathin sections were examined in voluntary ejection of poison in R. guttatus. Taking advantage of a LEO 906E microscope.

J. Exp. Zool. PAROTOID MACROGLANDS AND DEFENSE IN TOADS 3

Figure 1. (a) Adult specimen of Rhinella marina. The arrow indicates the parotoid pores. (b) Manual compression upon the parotoid of R. marina mimetizing the pressure exerted by the bite of a predator. (c) Adult specimen of Rhaebo guttatus. The arrow indicates the parotoid pores. (d) Voluntary ejection of poison through the parotoid of R. guttatus.(e–g) Sequence of behavioral defensive postures of R. guttatus culminating with poison squirting upwards (g).

Skin Secretion HPLC system (20A Prominence, Shimadzu Co., Kyoto, Japan). The parotoid secretion from eight specimens of R. marina and R. Aliquots of 20 mL of the secretions were loaded in a C18 column guttatus was obtained by manual compression of the macrog- (ACE C18, 5 mm, 100 A, 250 mm 4.6 mm) in a two‐solvent lands (Fig. 1b) using a manually operated pressure gauge to system: (A) TFA/H2O (1:1,000) and (B) TFA/acetonitrile (ACN)/H2O register the minimum pressure necessary (kg/cm2) for the poison (1:900:100). The column was eluted at a constant flow rate of to be sprayed. Due to their highly viscous nature, the poisons 1 mL min1 with a 0–100% gradient of solvent B over 20 min, after were extracted with 0.1% trifluoroacetic acid (TFA)/H2O a 5 min isocratic elution with 0% of solvent B. The HPLC column containing 5% acetonitrile. The solution was sonicated for eluates were monitored by a Shimadzu SPD‐M20A PDA detector 5 min. Alternatively, the crude secretion was diluted in PBS, pH scanning from 200 to 500 nm (1 nm steps). 7.4, sonicated and the protein concentration was estimated by The online mass spectrometry analyses were performed in an the BCA assay. ESI mass spectrometry (LCQDuo™, ThermoFinnigan, San Jose, CA USA), equipped with a nanospray source and connected to a nano Biochemical Analyses HPLC system (UltiMate, LC Packings/Dionex, Sunnyvale, CA The skin secretions were analyzed by reversed‐phase high‐ USA), coupled to a C18 nanocolumn. The samples were deposited performance liquid chromatography (RP‐HPLC) using a binary into autosampler and 5 mL sample aliquots were injected at a

J. Exp. Zool. 4 MAILHO‐FONTANA ET AL. constant flow rate of 1 mL min1. The spray voltage was kept at Statistical Analyses. Results are presented as mean standard 1.8 kV, the capillary voltage at 46 V, the capillary temperature at error of mean (SEM). Statistical evaluation of data was carried out 180°C, and the tube lens offset was kept at 5 V. MS spectra by Student's t‐test or repeated measures two‐way ANOVA acquired under positive mode and collected in the 50–2,000 m/z followed by Bonferroni or Tukey post‐test (GraphPad Prism 5, range. Instrument control, data acquisition and data processing GraphPad Software, Inc., La Jolla, CA USA). Differences of results were performed using the Xcalibur Suite. were considered statistically significant when P 0.05. In order to assess the protein contents of the extracts, a 12% polyacrylamide gel electrophoresis, containing sodium dodecyl sulfate (SDS–PAGE), was performed, in reducing conditions, RESULTS according to Laemmli ('70). Defensive Behavior When stimulated in their natural environment, both species Biological Assays showed typical defensive behavior of toads, consisting of inflating Lethality. Lethal activity was evaluated according to Finney ('71). the lungs, making snorting characteristic sounds, leaning the body Briefly, the poison was dissolved in phosphate‐buffered saline toward the stimulus, and exposing the parotoids (Fig. 1a, c, and e). (PBS), pH 7.4 and different doses were orally (7, 10, 14, 20, or R. guttatus, in particular, quickly opens and closes the mouth and 28 mg) or intraperitoneally (IP) (2.7, 4, 6, 9, or 13.5 mg) the eyes, and when directing one of its parotoids toward the administered in mice (n ¼ 6 per dose). A mouse control group aggressor, lowers the scapular girdle, turns the body sideways and, was injected with PBS, pH 7.4. Death was evaluated 48 or 96 hr with mouth and eyes closed, ejects the poison (Fig. 1d–g). Twenty‐ after IP or oral administration, respectively. one specimens of R. guttatus were stimulated to perform the defensive display; poison ejection occurred in approximately 57% Edema. Groups of mice (n ¼ 6 per group) had their right hind paw of the individuals. While displaying the behavior, the animals measured by a plethysmometer (7141 Plethysmometer—Ugo seem to choose which of the two parotoids will be used for ejection Basile, Comerio, Italy) before receiving an intraplantar injection and in which direction the poison jets will be launched. The jets are of five different doses (1,620, 540, 180, 60, or 20 mg, diluted in directed upwards and can reach distances greater than 2 m. The 30 mL PBS) of the poison of both toad species. A mouse control main target of the jets seems to be the face, but most of them are group was injected with PBS, pH 7.4. The results were expressed as dispersed in the air. The poison is yellowish and sticky, having a the difference between the measure of the displaced volume (mL) characteristic odor, like that of trampled grass. Held manually, the of the experimental paws in different time intervals (0.5, 1, 2, 4, 24, toads maintain the lungs inflated but stop ejecting the poison, 48, and 72 hr) and the displaced volume of the same paws in the which often continues to drip down from the surface of the initial time. parotoids. In R. marina, the minimum pressure required for the parotoid Estimation of Nociceptive Activity. To detect the nociceptive poison to be sprayed was 0.588 0.080 kg/cm2. On the other activity, groups of mice (n ¼ 6 per group) were injected in the right hand, R. guttatus required only 0.259 0.032 kg/cm2 kg to be hind paw with 30 mL of PBS containing different doses (20, 40, 80, released, a pressure approximately two times lower (P 0.001) 160, or 320 mg) of the poisons of both toad species. Mice were when compared to R. marina. placed individually under glass funnels on a mirror. Following, the time of reactivity of the animals licking or biting the injected foot Morphological Observations of the Skin and of the Parotoids was measured, in seconds, during 30 min of experimental visual The skin of R. marina is similar to that of other toads with many evaluation (Hunskaar et al., '85). Animals injected only with PBS warts and other small projections, giving a rugous aspect to the were used as negative controls. animal (Figs. 1a and 2a). In contrast, a very soft and smooth skin covers the whole body of R. guttatus (Figs. 1c and 2b). Hemorrhagic and Necrotic Activity. The hemorrhagic activity of Anatomical observation showed that in R. marina, as in other the poison of both toad species was verified, according to the toads, the parotoid is completely loose from the tissues beneath it modified method described by Kondo et al. ('60). Briefly, 20, 200, and is easily dissected and removed from the animal. Contrasting, and 1,000 mg of the poisons diluted in 0.2 mL of PBS were injected in R. guttatus part of the floor of the parotoid is attached to the (intradermic via) in the dorsal skin of mice (n ¼ 3 per dose). Two scapula by an area of adhesion located in the extreme of the hours after injection, animals were euthanized, and the skin in the external side of the parotoid (Fig. 2c–f). Histological sections show area of injection was removed to determine hemorrhagic spots on that a bundle of collagenous fibers, running parallel and between skin internal face. Control animals received only PBS. For the the scapula and the parotoid strongly attaches to this specific area, assessment of the necrotic activity, the same procedure was converges (Fig. 2e and f). repeated; however, animals were euthanized 72 hr after poison The dorsal skin of both R. guttatus and R. marina is covered by a administration. slightly pigmented epidermis, which in histological sections shows

J. Exp. Zool. PAROTOID MACROGLANDS AND DEFENSE IN TOADS 5

Figure 2. (a) Histological section of the dorsal skin of Rhinella marina. (b) Histological section of the dorsal skin of Rhaebo guttatus. Hematoxylin–eosin staining. (c) Schematic drawing representing a dissected parotoid and its associated muscles and bones. Note the adherence area (Aa) in the parotoid floor. (d) Histological section through the scapular girdle of R. guttatus showing the adherence area between the parotoid (P) and the outermost area of the scapula (Sp). Hematoxylin–eosin staining. (e) Detail of the adherence area of d. The arrows point to the close connection between the parotoid (P) and the scapula (Sp). Hematoxylin–eosin staining. (f) An equivalent histological section of e, observed under polarized light. The dense collagen fibers appear in red. The arrows point to the close connection between the parotoid and the scapula. Picrosirius staining. Legends: Ca, calcified dermal layer; E, epidermis; Dm, depressor mandibulae muscle; Ds, dorsalis escapulae muscle; g, granular gland; Ld, latissimus dorsi muscle; Sc, stratum compactum; Ss, stratum spongiosum;(), mucous gland.

to be constituted by about seven cell layers (Fig. 2a and b). In the layer, which is present in the whole dorsal skin, is abruptly dermis between the stratum spongiosum and the stratum interrupted around the parotoid, where it is completely absent compactum there is a calcified layer that is proportionally much (Fig. 3b). thicker in R. marina, especially in the parotoids, in which it is The analysis of histological serial sections showed that in the very robust and dense, being interrupted only in the alveolar parotoids of R. marina the alveolar plugs are constituted by a very ducts (Fig. 3a). In contrast, in R. guttatus the calcified dermal dense epithelial tissue compacted between the medial and the

J. Exp. Zool. 6 MAILHO‐FONTANA ET AL.

Figure 3. (a) Histological section of Rhinella marina parotoid macrogland. Observe the thick calcified dermal layer (Ca) stained in black. von Kossa histochemistry. (b) Histological section of Rhaebo guttatus parotoid macrogland. Observe that the thin calcified dermal layer (Ca) of the dorsal skin, stained in black, disappears as it approaches the parotoid. von Kossa histochemistry. (c). Histological section of R. marina parotoid focusing the epithelial plug (Pl) of an alveolus (Al). Note that the plug is long, dense and deep in relation to the surface of the skin. Hematoxylin–eosin staining. (d) Histological section of R. guttatus parotoid focusing the epithelial plug (Pl) of an alveolus (Al). Note that different from R. marina, the plug is short, have a slit in the center (arrow), and reaches the skin surface. Hematoxylin–eosin staining. (e) SEM of a R. marina parotoid pore, with a slit shape. The arrows point to the pores of the accessory glands. Note that the surface of the skin shows many protuberances (arrowheads). (f) SEM of a R. guttatus parotoid pore, with a round shape. It is possible to observe the epithelial plug (Pl) reaching the surface. The arrows point to the pores of the accessory glands. Legends: Al, alveolus, D, dermis; g, superficial glands of the parotoid; (), accessory glands.

J. Exp. Zool. PAROTOID MACROGLANDS AND DEFENSE IN TOADS 7 innermost portion, in which it is in close contact with the TEM showed that, in both species, each syncytial glandular unit poisonous secretion (Fig. 3c). Differently, in the parotoids of R. of the parotoids inside the alveoli is surrounded by a monolayer of guttatus, the plugs are characterized by a large crack in the center myoepithelial cells (Figs. 4a and b). These cells show elongated that tapers towards its inner portion (Fig. 3d). As a consequence, in nuclei and cytoplasm full of filaments longitudinally arranged, this species only a short layer of ductal epithelial cells separates similarly to smooth muscle cells (Fig. 4c). On their outer side, in the poison from the outside. close contact with the dermis, they lay on a well‐defined basal SEM showed that the superficial morphology of the skin and of lamina. In transverse sections, it is possible to observe that the the pores is quite different between the two species. In R. marina cells are connected to each other and to the syncytium by the skin surface is full of small corneous projections and the pores interdigitations and desmosomes. Nerve cells are also present are externally observed as slit‐shaped structures located in among the cells. In both internal and external cytoplasm depressions in the skin (Fig. 3e). Around them, pinholes, also membranes, a large number of pinocytosis vesicles are visible. slit‐shaped, are seen representing the pores of the differentiated The only difference between both species seems to be in the mucous glands. In R. guttatus, the skin surface of the parotoid is amount of clusters of glycogen, which seem to be more abundant very smooth and the pores appear as round structures located in the myoepithelial cells of R. guttatus (Fig. 4d). more superficially than in the R. marina (Fig. 3f). The epithelial plugs do not completely obstruct the pores, indicating they are not Biochemical Characterization of the Parotoid Secretion fully adhered to the skin lining the duct. Around each pore, other The parotoid poisons of R. marina and R. guttatus showed similar pores of much smaller proportion are seen, corresponding to the electrophoretic profiles (Fig. 5), especially in the major proteins: differentiated mucous glands; these are much rounder when 116, 55, and 30 kDa. While between 116 and 55 kDa the compared to those of R. marina. profiles are very similar, in the range of 30–55 kDa there are a few

Figure 4. TEM. Myoepithelial cells (My) of Rhinella marina parotoid in longitudinal view (a) and oblique view (b), showing the dense cytoplasm full of actin fibrils, mitochondria (m) and pinocytosis vesicles (arrowheads). The area of contact between myoepithelial cells and the syncytium (Sy) is full of interdigitations (#). Nervous endings () are also found in this area and among the cells. Myoepithelial cells (My) of Rhaebo guttatus parotoid in oblique view (c) and transversal view (d). Differently from R. marina, in this species we noticed abundant glycosomes (Gl) within the cytoplasm. The arrows point to desmosomes between the myoepithelial cells. Legends: N, nucleus; Sy, syncytium; (), nervous ending.

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Evaluation of Some Biological Activities Both poisons when injected intraperitoneally at high doses were lethal. However, the poison from R. marina proved to be more toxic than that from the R. guttatus (Table 1). The experiment of oral lethality did not allow obtainment of results for

calculation of LD50, since the highest dose of R. marina poison used was able to kill only one animal. However, all animals after oral application of the poison showed clear signs of poisoning, including seizures and alternating states of prostration and excitement. The poison of the parotoids of R. marina did not induce edema at any of the doses tested, with values similar to the group injected only with PBS (Fig. 7a). On the other hand, the poison from the parotoids of R. guttatus was able to induce edema at all tested doses (Fig. 7b). The climax of the edema was reached in 4 hr, lasting 72 hr at the highest dose used. Poisons from both toads are capable of inducing nociception in mice (Fig. 7c). However, the poison of R. guttatus induces about four times more reactivity when compared to R. marina. None of the poisons were able to induce, by the methods employed, Figure 5. SDS–PAGE of the parotoid poisons of Rhinella marina hemorrhage or necrosis. (Rm) and Rhaebo guttatus (Rg). The numbers (kDa) at the right correspond to the molecular mass markers. DISCUSSION To avoid confusion with other cutaneous skin glands, in this article, when referring to parotoids, we have used the term “macroglands” instead of “glands”, as these structures are clearly bands that are unique for each poison. In the region of 14 kDa a multiglandular organs (Toledo and Jared, '95; Jared et al., 2009). conspicuous and exclusive band of R. guttatus mark the most Furthermore, following the correct etymology, we opted for the evident difference between the poison of both species. term “parotoid” instead of “paratoid” or “parotid” (Cannon and The chromatographic profiles obtained from the poisons of the Palkuti, '76; Tyler et al., 2001). two toads are similar (Fig. 6a–d). Both are clearly made up of two The mechanism of action of the parotoids in bufonid passive groups of components, biogenic amines, eluted between 15 and defense was first studied by Jared et al. (2009), taking as model the 18 min, and bufadienolid steroids, eluted between 22 and 27 min. toad Rhinella jimi, a species of the Brazilian semiarid region Analysis by offline mass spectrometry (Fig. 6a and c) indicated (Caatinga), belonging to the R. marina group (Pramuk, 2006). In the presence of serotonin and its derivatives (dehydrobufotenin this study, Jared et al. (2009) showed that this species is unable to and N‐methyl‐serotonin) in the poison of both species. Addition- voluntarily eject its poison and suggest that, due to the similarities ally, the steroids marinobufagin, telocinobufagin, and bufalin in the defensive behaviors and in the structure of the parotoids were identified only in the poison of R. marina while resin- among toads, such characteristic must be present in the whole obufagin (bufadienolid steroids) was identified only in the poison group. As a consequence, poisoning by a toad occurs only when of R. guttatus. The biogenic amines comprise the group of the predator attacks and directly bites the toad's parotoids. substances representing the greatest peaks in both poisons. The However, Jared et al. (2011) found that, contrary to the typical dehydrobufotenin amine is most abundant in the poison of R. passive defensive behavior of other toads, R. guttatus is able to marina while serotonin is the component with the highest peak voluntarily squirt poison jets from their parotoids at great intensity in R. guttatus. distances. Our data show that the defensive behavior of R. marina, Through analysis by online mass spectrometry (Fig. 6b and d) as well as the morphology of its parotoids, follow the same pattern we identified the amines dehydrobufotenin, bufotenin, serotonin already described for other Rhinella toads, also favoring the and N‐methylserotonin, and the steroids helebregenin, telocino- predator's self‐poisoning, while R. guttatus show behavioral and bufagin, marinobufagin, resinobufagin, and bufalin. The chro- morphological peculiarities that can explain their ability to matographic profile generated by mass detector was similar for voluntary eject poison from their parotoids. Also, we found both poisons, as was the chromatogram generated by RP‐HPLC biochemical and pharmacological differences in the poison of the analysis. In addition, in R. guttatus poison, some of the molecular two species that are suitable with the peculiarities of their mass did not match with any molecule described so far. defensive mechanisms.

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Figure 6. (a and b) Chromatographic profiles of Rhinella marina parotoid poison. Off‐line mass spectrometry (a); on‐line (LC‐MS/MS) (b). (c and d) Chromatographic profiles of Rhaebo guttatus parotoid poison. Off‐line mass spectrometry (c); on‐line (LC‐MS/MS) (d). Legends: 1, serotonin; 2, dehidrobufotenin; 3, N‐metil‐serotonin; 4, marinobufagin; 5, telocinobufagin; 6, bufalin; 7, resinobufagin; 8, bufotenin; 9, helebregenin.

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Table 1. Lethality induced by the poison extracted from the parotoids of Rhinella marina (Rm) and Rhaebo guttatus (Rg).

a LD50 IP Rm Rg mg/animal 4.35 5.55 mg/kg 217.5 277.5

aIntraperitoneal lethal dose 50% estimated by Probitos method (Finney, '71).

R. guttatus (Schneider, 1799) was described over 210 years ago and there is virtually no information on its biology and natural history. Regarding its unusual defensive mechanism, it is expected that the morphological and chemical arsenal used in its defense might be different from other toads with passive defense. Among our findings, the first that seems to contribute to the ability of R. guttatus to voluntarily eject poison is the association of its parotoids with structures related to the movement of the scapula, which was not seen in R. marina. Thus, the behavior of lowering the scapular girdle seems to be important to increase the alveolar pressure inside the parotoid. This pressure, together with that exerted by the inflated lungs and coupled to the unique morphological characteristics of the parotoids, must be sufficient to the voluntary ejection of the poison. On the other hand, the examination of the parotoids of R. marina and R. guttatus revealed many structural similarities, which are also shared by other bufonids that were already studied (Hostetler and Cannon, '74; Toledo et al., '92; Almeida et al., 2007; Felsemburgh et al., 2009; Jared et al., 2009). In all toads these macroglands are composed of juxtaposed secretory alveoli with a characteristic honeycomb‐like structure. The histology shows that in all toads, including R. guttatus, exclusive epithelial plugs made up of several layers of epidermal cells always block the alveolar ducts. Such plugs retain the internal pressure of the alveoli and are thus related with poison expelling (Jared et al., 2009). It was exactly in the plugs that we found one of the most evident morphological differences between the parotoids of the two toads. In R. guttatus the plugs are short and practically reach the surface of the skin, and have a large slit in the center that make then more susceptible to disruption, requiring lower alveolar pressure for the poison to be released, and favoring the voluntary mechanism of poison ejection. In contrast, in R. marina, the deeper position of the plugs in relation to the pores and the total ductal obstruction Figure 7. (a and b) Edematogenic activity induced by the parotoid may require higher internal alveolar pressures for poison poison of Rhinella marina (a) and Rhaebo guttatus (b). (c) expelling, which necessarily is dependent of the strength of a Nociceptive activity of the parotoid poison of R. marina (left) and R. predator's bite. In fact, we found that R. marina requires a manual guttatus (right). Legends: (), significant statistical difference pressure twice as higher than the pressure applied to the R. versus control; (#), the same dose of poison. guttatus parotoids. Thus, our data reinforce the idea that the morphology of the plug is an important factor in poison ejection. Since R. guttatus is a “non‐conventional” toad in relation to the mechanism of chemical defense, we also investigated possible

J. Exp. Zool. PAROTOID MACROGLANDS AND DEFENSE IN TOADS 11 ultrastructural differences in the myoepithelial cells enveloping toad (Burnett, '97; Doody et al., 2006; Griffiths and McKay, 2007; the parotoid alveoli that could justify voluntary poison squirting. Crossland et al., 2008; Letnic et al., 2008; O'Donnell et al., 2010). When compared to R. marina, except for the apparent greater Our results confirmed the toxicity of R. marina poison, showing amount of glycogen present in the myoepithelial cells of Rhaebo that in mice its lethality is higher than that shown by R. guttatus. guttatus, there is no other significant structural fact supporting an Surprisingly, however, the parotoid poison of R. guttatus, despite effective participation of these cells in poison release. Many causing low lethality, causes significant levels of inflammation studies (Benson and Hadley, '69; Mcafee, '70; Delfino et al., '82) and nociception, symptoms that seem to be very adequate to act at have shown that in amphibian skin the catecholamines a distance, at the moment the poison jets reach the mucosa of the (particularly noradrenaline) can induce a rapid discharge of the aggressor that is probably discouraged to proceed in the attack. granular glands contents on the skin surface by the contraction of The non‐lethality of this poison also seems more adequate for a myoepithelial cells (Hoffman and Dent, '77). However, in the mode of defense in which a large part of the parotoid discharge is parotoids of toads, which depend on external stimulation for lost into the environment, before reaching the attacker. In poison ejection, the myoepithelial cells have been more associated contrast, in R. marina defense, the attacker receives the entire with the need of constant homogenization of the secretion in the discharge of the lethal poison, directly reaching its mouth. interior of the syncytia, due to their large volume (Jared et al., Recently Peredero et al. (2012) reported the effects of R. guttatus 2009). poison on a domestic dog. When approaching the toad, the dog Another significant morphological feature found in the received squirts of poison that hit its eyes and ear. Edema, parotoids of R. guttatus, is the exclusively absence of a calcified erythema, apparent pain and red eyes were observed shortly after dermal layer in the parotoids, although present in the rest of the and persisted for nearly one hour, even with the affected area body skin. The calcified layer of the anurans skin, commonly being thoroughly washed. This report confirms our experimental related to water economy, is a distinctive feature of the skin of this results and demonstrates that our findings are entirely consistent group of amphibians (Elkan, '68, '76; Toledo and Jared, '93). It with R. guttatus natural defense dynamics. usually covers the entire body skin and is located deep in the According to Daly et al. ('93) amine dehydrobufotenin has dermis, below the layer of cutaneous glands (Schwinger convulsive activity. Since this compound occurs in large et al., 2001; Brito‐Gitirana and Azevedo, 2005; Felsemburgh quantities in the secretion of the parotoids of both R. marina et al., 2009; Jared et al., 2009). In bufonids, the calcified layer is and R. guttatus, the convulsions we observed in mice are probably well developed and creates a type of armor that hinders water loss due to the action of this biogenic amine. We also detected in terrestrial environments and in the open areas where these bufotenin in R. marina, a compound that presents hallucinogenic animals commonly inhabit (Elkan, '68, '76; Toledo and Jared, '93, action and, according to Forsström et al. (2001), has also been '95; Navas et al., 2005; Jared et al., 2009). Differently from the rest detected in the urine of schizophrenic patients. Such hallucino- of the skin, in the parotoids of toads, including R. marina, the genic activity would explain the alternation of prostration with calcified layer seems to be exceptionally well developed and periods of intense agitation of the mice injected with the poison of allocated above the glandular alveoli, very close to the epidermis this toad. In addition, the serotonin we found in the poison of both (Toledo et al., '92; Almeida et al., 2007; Felsemburgh et al., 2009; toads is a vasoconstrictor compound and alters thermoregulation Jared et al., 2009). According to Baldwin and Bentley ('81) the and the motor function of the gastrointestinal tract (Schwartz calcified layer must act as a mechanical barrier on the skin, et al., 2007), symptoms that are common in poisoning by toads exerting a protective function and therefore hardening the skin (Pineau and Romanoff, '95; Sakate and Lucas de Oliveira, 2000). and making it less malleable. The absence of a calcified dermal Many other animals, including arthropods, at least one layer in the parotoids of R. guttatus seems, therefore, to be very salamander (Salamandra salamandra), some snakes, lizards, convenient for this species, conferring higher flexibility to these and skunks use sprays of chemicals as defense, directing them macroglands to squirt the poison without the need of an external at predators (Eisner, '70; Edmunds, '74; Rosenberg and Russell, '80; pressure, as is the case of the other toads. Brodie and Smatresk, '90). According to Brodie and Smatresk ('90) The sensitivity of various vertebrates, including humans, to the the obvious advantage of this strategy is that the prey with the poison from bufonids is well known (Sakate and Lucas de ability to spray chemicals can repel the predator before physical Oliveira, 2000; Doody et al., 2006; Griffiths and McKay, 2007; assault or injury. Such defensive behaviors were probably Keomany et al., 2007; Letnic et al., 2008). Cardiotoxic activities are mechanisms favored by natural selection to speed up predators' described, among other convulsive and hallucinogenic effects learning. This seems to be the case of R. guttatus that, when (Hanson and Vial, '56; Toledo and Jared, '95; Schwartz et al., 2007) compared to the after‐biting chemical defensive system used by R. as well as symptoms such as hiccups, dysentery, fainting, and marina, shows immediate benefits. The aggressive and imposing deliriums when the poison is ingested (Toledo and Jared, '95). The initial posture starts predator's intimidation. Parotoid voluntary high lethality of the poison of R. marina has been observed in compression makes possible for the potential predator to Australia on reptiles and native mammals that try to feed on this experience the poison effects even at a distance; with its non‐

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