The skin secretions are more toxic than the venom of the pitvipers, Bothrops

Two Brazilian frogs have bony spines around the margin of their skull

Venomous animals have toxins associated with delivery mechanisms that can introduce the toxins into another animal []. Although most amphibian species produce or sequester noxious or toxic secretions in the granular glands of the skin to use as antipredator mechanisms [], amphibians have been considered poisonous rather than venomous because delivery mechanisms are absent. The skin secretions of two Brazilian hylid frogs (Corythomantis greeningi [] and Aparasphenodon brunoi) are more toxic than the venoms of deadly venomous Brazilian pitvipers, genus Bothrops []; C. greeningi secretion is 2-fold and A. brunoi secretion is 25-fold as lethal as Bothrops venom. Like the venoms of other animals, the skin secretions of these frogs show proteolytic and fibrinolytic activity and have hyaluronidase, which is nontoxic and nonproteolytic but promotes diffusion of toxins. These frogs have well-developed delivery mechanisms, utilizing bony spines on the skull that pierce the skin in areas with concentrations of skin glands. C. greeningi has greater development of head spines and enlarged skin glands producing a greater volume of secretion, while A. brunoi has more lethal venom. C. greeningi and A. brunoi have highly toxic skin secretions and an associated delivery mechanism; they are therefore venomous. Because even tiny amounts of these secretions introduced into a wound caused by the head spines could be dangerous, these frogs are capable of using their skin toxins as venoms against would-be predators.

Results and Discussion

50 ) of A. brunoi secretion was as follows: head, 3.12 μg; body, 4.36 μg. The LD 50 of C. greeningi was as follows: head, 51.94 μg; body, 49.34 μg. Skin secretion of both species induced edema in mice at all tested doses, reaching the highest values at a dose of 8 μg after 30 min for A. brunoi and after 60 min for C. greeningi ( 6 Lynch J.D.

Ramírez M.A.V. Lista preliminar de especies de anuros del departamento del Guainía, Colombia. Figure 1 Head Spines of Aparasphenodon brunoi and Corythomantis greeningi Show full caption (A and B) Adult frogs A. brunoi (A) and C. greeningi (B). (C and D) Co-ossified skulls of A. brunoi (C) and C. greeningi (D); arrowheads point to occipital region. (E and F) Higher magnification of the rostral margin of the skull of A. brunoi (E) and C. greeningi (F). Figure 2 Edematogenic and Nociceptive Activity of Venoms Show full caption ∗) versus same dose of venom (#). See also (A–C) Edematogenic activity of A. brunoi (A) and C. greeningi (B) venoms. Nociceptive activity of venoms of both species (C). Two-way ANOVA followed by Tukey post-test. Differences between results were considered statistically significant when p ≤ 0.05. All experiments utilized six Swiss male mice per dose per group, weighing 18–20 g. Bars represent means, and vertical lines show SEM. Significant statistical difference versus control () versus same dose of venom (#). See also Figure S1 In order to demonstrate that an organism is venomous, it is necessary to determine that toxins are both present and associated with a delivery mechanism. We have determined that the skin secretions of Aparasphenodon brunoi ( Figure 1 A) and Corythomantis greeningi ( Figure 1 B) are highly toxic. Secretions from the head or body of both species were lethal to mice when injected intraperitoneally in microgram quantities. The lethal dose 50 (LD) of A. brunoi secretion was as follows: head, 3.12 μg; body, 4.36 μg. The LDof C. greeningi was as follows: head, 51.94 μg; body, 49.34 μg. Skin secretion of both species induced edema in mice at all tested doses, reaching the highest values at a dose of 8 μg after 30 min for A. brunoi and after 60 min for C. greeningi ( Figures 2 A and 2B ). There was persistent edema 72 hr after injection, particularly at doses of 32 μg and 8 μg in both species, and for C. greeningi even at a dose of 0.5 μg. Secretions of both frogs induced nociception in mice, regardless of dose ( Figure 2 C). The skin secretion of a related species, A. venezolanus, has been reported to cause pain and visual impairment in humans [].

1 Bucherl, W., and Buckley, E., eds. (1969, 1971). Venomous Animals and Their Venoms, Volumes 1–3 (New York: Academic Press). 7 Fry B.G.

Roelants K.

Champagne D.E.

Scheib H.

Tyndall J.D.A.

King G.F.

Nevalainen T.J.

Norman J.A.

Lewis R.J.

Norton R.S.

et al. The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms. 8 Tu A.T. Venoms: Chemistry and Molecular Biology. 8 Tu A.T. Venoms: Chemistry and Molecular Biology. 9 Tu A.T.

Hendon R.R. Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor. 9 Tu A.T.

Hendon R.R. Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor. The electrophoretic profiles of proteins in the secretions from the head and body of both species showed a number of similarities, but there were some bands unique to each species ( Figure S1 ). The differences between the two species were clear in the regions between 12 and 37 kDa and between 45 and 120 kDa. Additionally, there were unique bands in the head secretion of each species at about 60 kDa. The secretion of A. brunoi showed a greater diversity of bands with enzyme activity than did the secretion of C. greeningi ( Figure S1 ). The secretions showed proteolytic activity typical for animal venoms []. Gelatinolytic and caseinolytic activities were detected exclusively in the body secretion of A. brunoi. Fibrinogenolytic activity was detected in the body secretion of A. brunoi and in both body and head secretions of C. greeningi. A common single band showing hyaluronidase activity was detected in all samples except the body secretion of A. brunoi. Hyaluronidase is present in the venoms of all major families of venomous snakes [] and Gila monsters []; it is nontoxic and nonproteolytic but acts as a spreading factor to allow diffusion of toxins []. We are unaware of reports of hyaluronidase in other amphibian skin secretions.

4 Jared C.

Antoniazzi M.M.

Navas C.A.

Katchburian E.

Freymüller E.

Tambourgi D.V.

Rodrigues M.T. Head co-ossification, phragmosis and defense in the casque-headed tree frog Corythomantis greeningi. Figure 3 Spines Pierce the Skin in A. brunoi and C. greening Show full caption (A) Live specimen of C. greening. (B–E) Scanning electron microscopy (SEM) of the rostral area and skin glands of A. brunoi (B and C) and C. greeningi (D and E). Spines (∗) penetrate the skin through regions with a high number of granular gland pores (arrows) on the skin surface. (D) Tangential and superficial section through the dorso-lateral region of the head, near the upper jaw, showing spines (∗) surrounded by granular glands (g). (E) Higher magnification of a region equivalent to (D) showing connective tissue surrounding each gland. Figure 4 Head Spine and Skin Gland Histology Show full caption (A) Median sagittal section through the head of A. brunoi; arrows point to regions with major concentrations of glands. (B) Median sagittal section through the head of C. greeningi showing a high number of large granular glands. The asterisk indicates a spine almost reaching the skin surface. (C) High magnification of an area of co-ossification in the top of the head of A. brunoi. (D) High magnification of an area of co-ossification in the upper lip of C. greeningi. (A–D) Stained with hematoxylin-eosin. (E and F) Granular glands of A. brunoi (E) and C. greeningi (F) are rich in protein content (stained with bromophenol blue). Some cells of the mucous glands also show protein. ∗) indicates calcified dermis with spines. See also e, epidermis; d, dermis; g, granular gland; m, mucous gland; s, skull; asterisk () indicates calcified dermis with spines. See also Figure S1 The venom delivery mechanism in these frogs is in the form of spines on the head associated with the toxin-producing skin glands. The heads of both A. brunoi ( Figures 1 A, 1C, and 1E) and C. greeningi ( Figures 1 B, 1D, and 1F) are flattened and rough, with a co-ossified dermal stratum compactum and prominent occipital and labial spiny ridges ( Figures 3 A–3D and 4 A–4D). They have rostral projections that form a conspicuous protrusion similar to an upper lip, especially in C. greeningi []. The skulls of both species have numerous bony spines, enlarged in the nasal, jaw, and occipital regions ( Figures 1 C–1F). The rostral areas around the nostrils and in the superior lip ( Figures 4 A–4F) show more prominent spines, associated with concentrations of mucous and especially granular glands ( Figures 3 B, 3C, and 4 B–4D). The granular glands in the skin of the head are enlarged in C. greeningi (320 ± 14 μm in diameter and 524 ± 30 μm in height), in comparison with the granular glands on the body (123 ± 25 μm in diameter and 30 ± 20 μm in height). Head and body granular glands of A. brunoi are similar in size (121–129 μm diameter and 123–130 μm height) and are much smaller than the head glands of C. greeningi. However, the glands of the dorsal skin and head in both species are similar histochemically ( Figures 4 E and 4F).

In both species, spines pierce the epidermis in areas of the skin well supplied with granular glands ( Figures 3 A–3D, 4 C, and 4D). When restrained by hand, these frogs release a sticky secretion and flex the head, jabbing and rubbing the spines into the hand. Many of the spines pierce the skin and are coated with the skin secretion ( Figure 3 A). These frogs have an unusual ability to flex the head laterally and vertically, as compared to most other frogs, thereby facilitating contact between the spines in the rostral and posterior margin of the head and the hand grasping the frog. One of us (C.J.) was injured on the hand by the spines of C. greeningi while collecting frogs, causing intense pain radiating up the arm, lasting about 5 hr. This action should be even more effective on the mouth lining of an attacking predator.

50 of 94.8 μg in mice [ 5 Sanchez E.F.

Freitas T.V.

Ferreira-Alves D.L.

Velarde D.T.

Diniz M.R.

Cordeiro M.N.

Agostini-Cotta G.

Diniz C.R. Biological activities of venoms from South American snakes. In conclusion, we have demonstrated that the Brazilian frogs C. greeningi and A. brunoi produce highly toxic skin secretions, have an active delivery mechanism, have bony spines that pierce dense beds of toxin-producing skin glands, and should thus be considered venomous. The toxicity of the skin secretion of A. brunoi is truly impressive, being 25-fold higher than that found in the deadly venomous pitviper genus Bothrops, which averages an LDof 94.8 μg in mice []. Interestingly, the toxicity of C. greeningi, while still substantial (nearly twice that of Bothrops), is less than that of A. brunoi; however, C. greeningi has greater development of head spines and enlarged skin glands producing a greater volume of secretion.