Zoomorphology (2017) 136:219–224

DOI 10.1007/s00435-017-0344-0

ORIGINAL PAPER

Developmental morphology of granular skin glands in pre-

metamorphic egg-eating poison frogs
Jennifer L. Stynoski1   · Lauren A. O’Connell2 

Received: 8 November 2016 / Revised: 24 January 2017 / Accepted: 27 January 2017 / Published online: 20 February 2017
© Springer-Verlag Berlin Heidelberg 2017

Abstract  Parents in many taxa, including insects, mol- Keywords  Alkaloid · Ontogeny · Poison gland ·
luscs, fish, snakes, and amphibians provision chemical Provisioning · Tadpole
defences, such as peptides, steroids, or alkaloids to their
offspring to reduce the risk of predation. In most cases,
those defences are transferred to offspring in the egg and Introduction
gradually diminish throughout the larval period. Adult poi-
son frogs sequester alkaloid-based defences from arthro- Parents in many taxa reduce predation of young by provi-
pod prey in granular skin glands. In at least one poison sioning offspring with chemical defences (Hutchinson et al.
frog, Oophaga pumilio, mother frogs intermittently feed 2008; Stynoski et al. 2014a; Santos et al. 2016 and therein).
tadpoles until metamorphosis with nutritive eggs contain- While this behaviour and its associated chemical ecol-
ing those alkaloid-based defences. However, alkaloids are ogy is well studied in some invertebrates (e.g., Dussourd
not detected in tadpoles until they reach the middle stages et al. 1988; Williams et al. 2011), less is known about ver-
of larval development. Here, we investigate the histology tebrates that endow eggs with chemical defences, such as
of a developmental series of O. pumilio tadpoles to deter- some fish (Itoi et al. 2013), snakes (Hutchinson et al. 2008),
mine whether their ontogenetic alkaloid profile coincides salamanders (Hanifin et  al. 2003), and frogs (reviewed
with granular gland development. Our findings suggest that in Gunzburger and Travis 2005). Even less is understood
alkaloid sequestration in tadpoles is delineated by the dif- about more complex vertebrate systems in which chemi-
ferentiation of rudimentary granular skin glands in epithe- cal provisioning continues after hatching or birth, such as
lial tissue. The timing of differentiation of granular glands in the strawberry poison frog (Oophaga pumilio; Stynoski
in this species coincides with other anurans. Thus, provi- et al. 2014a). A complete understanding of the processes of
sioning of chemical defences to offspring is likely con- sequestration, transport, and storage of diet-based chemi-
strained by developmental timing of derived structures that cals in parents and offspring will require ecological, mor-
can effectively store those toxic or noxious compounds. phological, genomic, and functional perspectives. Such an
understanding will play a key role in elucidating the evolu-
tion of chemical defence and the mechanisms behind the
management of alkaloid compounds with important bio-
Electronic supplementary material  The online version of this
logical functions as well as real-world applications (e.g.,
article (doi:10.1007/s00435-017-0344-0) contains supplementary
material, which is available to authorized users. medicines and agricultural toxins).
The unique skin of amphibians is filled with mucous,
* Jennifer L. Stynoski granular, and/or lipid glands (Toledo and Jared 1995; Angel
[email protected]
et  al. 2003; Delfino et  al. 2010; Chammas et  al. 2015).
1
Department of Biology, Colorado State University, 200 West Granular (serous, poison) glands synthesise or sequester
Lake St., Fort Collins, CO 80524, USA an incredible diversity of compounds that serve as defence
2
FAS Center for Systems Biology, Harvard University, 52 against predators and pathogens (Saporito et al. 2012; San-
Oxford Street, Cambridge, MA 02138, USA tos et  al. 2016; Mina et  al. 2015). Chemical defences are

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220 Zoomorphology (2017) 136:219–224

especially important to the survival of amphibians during suggested that this ontogenetic alkaloid profile may reflect
the juvenile and adult phases and in some cases the egg temporal modulation of alkaloid provisioning by mother
phase (Gunzburger and Travis 2005; Saporito et  al. 2012; frogs or developmental shifts in tadpole sequestration abil-
Santos et  al. 2016). However, tadpoles are thought to be ity in the skin or elsewhere (Stynoski et al. 2014a). In this
less chemically defended, because they lack both yolk and study, we investigate the latter hypothesis.
granular glands in which to store toxic or noxious com- Juvenile O. pumilio have small granular glands that
pounds (reviewed in Gunzburger and Travis 2005). Stud- increase dramatically in diameter at sexual maturity (Sapo-
ies on the development of amphibian granular glands are rito et  al. 2010). In related dendrobatid species in which
limited (Angel et  al. 2003; Gunzburger and Travis 2005; alkaloid sequestration in detritus-eating tadpoles is not
Delfino et  al. 2010; Chammas et  al. 2015). In anurans, known (Dendrobates auratus, Angel et  al. 2003; Phyllo-
ectoderm-derived precursor cells of glands begin to aggre- bates bicolor; Delfino et al. 2010), rudimentary glands are
gate in pre-metamorphic stages (~30–38 of staging table by first seen in late pre-metamorphic stages. However, the lar-
Gosner 1960), form an alveolus containing early granules val morphology of granular glands has not been described
in the initial metamorphic stages (~38–40), and become in the obligatorily egg-feeding genus, Oophaga. Here, we
small adult-type glands with secretory product present dur- describe the timing of granular gland morphogenesis in
ing metamorphic climax (~41–46; see Fig.  1; Toledo and a developmental series of larval O. pumilio in an effort
Jared 1995). Glands migrate from the epidermis to the der- to determine whether the onset of alkaloid sequestration
mis during the terrestrial juvenile phase (Toledo and Jared known in this species coincides with gland ontogeny. In
1995). addition, because skin alkaloids are diet-based, changes
In O. pumilio, mother frogs visit larval offspring every in tadpole oral structures across development could affect
1–8 days across 6 or more weeks of development to provide their ability to ingest and sequester alkaloids. Therefore,
unfertilised eggs as food, without which offspring starve we also examine whether development of oral structures
(Brust 1993). Tadpoles of other species in this group, at is concurrent with granular gland maturation and alkaloid
least beyond the mid-larval stages, exhibit mouthparts that sequestration.
are specialized for an egg-based diet (Myers et  al. 1984;
Grant et  al. 2006). Recent work demonstrated that moth-
ers provision nutritive eggs with alkaloids derived from a Methods
diet of leaf-litter arthropods (Stynoski et al. 2014a), which
effectively defend offspring from some predators (Stynoski Oophaga pumilio tadpoles were reared by captive-bred
et  al. 2014b). Alkaloids were not detected in recently parents in tanks at Harvard University. Mother frogs
hatched tadpoles, but were detected in rising concentration were fed Drosophila flies and, therefore, were not pro-
and diversity after stage 34 (Stynoski et al. 2014a). It was visioning alkaloids to tadpoles. Seven tadpoles (stages

Embryo Premetamorphosis Prometamorphosis Metamorphic Climax Juvenile

Developmental
Stage: 1-25 26-30 30-35 36-37 38-40 41 42-45 46

1
Rana pipiens
2
Rana sylvatica
3
Xenopus laevis
4
Phyllomedusa hypochondrialis
5
Scinax nasica
6
Dendrobates auratus
7
Phyllobates bicolor
8
Rhinella granulosa

Oophaga pumilio

Fig. 1  Literature reports of timing of anuran granular skin gland sent). Stages reported according to Gosner (1960), following conver-
development, indicating the first stage examined that contained struc- sion from Nieuwkoop and Faber (1956) or Taylor and Kollros (1946)
tures. Early glands (grey bars) refer not only to a cluster of rudimen- staging tables when necessary using Table  2.1 in McDiarmid and
tary gland cells, but to an alveolar configuration. Adult-like glands Altig (1999). 1 Bovbjerg (1963), 2 Formanowicz and Brodie (1982)
containing granules of secretory product (black bars) generally 3 Reilly et al. (1994), 4 Delfino et al. (1998), 5 Terreni et al. (2003), 6
appear during or just prior to metamorphic climax (i.e., four legs pre- Angel et al. (2003), 7 Delfino et al. (2010), 8 Chammas et al. (2015)

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25, 25, 27, 32, 33, 40, and 41) were euthanised by appli- Discussion
cation of topical 20% benzocaine and fixed in 4% para-
formaldehyde in 1X phosphate buffered saline (PBS) We provide histological evidence that the developmen-
overnight at 4 °C. The following day, tadpoles were tal morphology of the granular skin glands in Oophaga
cryoprotected in 30% sucrose in 1X PBS with 0.01% pumilio coincides with the species’ ontogenetic alkaloid
sodium azide and stored at 4 °C for 5 days until shipment profile (Stynoski et al. 2014a). Here, we show that granu-
to Colorado State University for sample processing. lar skin glands first develop between tadpole stages 32
We verified tadpole stage and photographed each and 40 in Oophaga pumilio. Past work detected mater-
tadpole and its mouthparts using an Olympus stereo- nally derived alkaloids in tadpoles after stage 34 and in
scope, and then used the ImageJ 1.47t (NIH) software to increasing amounts up to stage 45, but not yet at stage 25
measure total length (Table  1). We flash froze tadpoles (Stynoski et al. 2014a). Therefore, it is likely that pre-met-
in isopentane and sectioned in the transverse plane on a amorphic morphogenesis of skin structures—rather than
cryostat at 18 μm. We stained tissue with toluidine blue modulation of alkaloid provisioning to nutritive eggs by
and counterstained with eosin. All tissue sections were mother frogs—delimits the onset of alkaloid sequestration,
examined with an Olympus microscope at 10-20X for although the alkaloid content of trophic eggs across tadpole
the presence of developing granular or other glands. development remains to be determined.
Tadpoles are thought to be most vulnerable during
metamorphic climax (stages 42–45) due to higher caloric
content, reduced swimming ability with four bulky limbs,
Results and energetic allocation to morphological transforma-
tion (Wassersug and Sperry 1977; Arnold and Wassersug
Tadpoles at stages 25–27 did not show evidence of 1978). Acquiring defensive chemicals prior to metamor-
glands, nor any skin structures resembling rudimentary phosis, whether via biosynthesis as in some other amphib-
granular glands or mesenchymal cell clusters, as in other ians or through sequestration of egg-derived alkaloids as in
studies (Toledo and Jared 1995; Angel et al. 2003; Del- O. pumilio (reviewed in Saporito et al. 2012; Santos et al.
fino et al. 2010; Chammas et al. 2015; Fig. 2). At stages 2016), should benefit metamorphosing tadpoles (Stynoski
32–33, tadpole epidermis contained some clustered et  al. 2014b). An additional row of keratodonts and a
mesenchymal cells, but no mature structures. At stages stronger and sharper upper jaw sheath appear concurrently
40–41, the epidermis contained numerous small granular with early granular glands (as seen in other maturing den-
glands with alveoli that were just beginning to migrate drobatid tadpoles; Myers et  al. 1984; Grant et  al. 2006).
to the dermis. The alveoli contained translucent granules Although these tadpoles eat eggs throughout the larval
as in Angel et al. (2003) (Fig. 3). period (Brust 1993; Stynoski 2009), these oral structures
Early (stage 25–27) tadpoles had a keratinised upper may aid late stage tadpoles in better tearing and anchor-
jaw sheath, but no keratodont rows (Fig.  4). Later tad- ing on to them (de Sousa et  al. 2014), perhaps increasing
poles (stage 32–41) possessed one posterior keratodont consumption of alkaloid-containing material once they can
row, the oral disc papillae were more elaborate and thick, sequester those compounds.
and the upper jaw sheath was larger and more serrated. Past studies have shown that alkaloid concentration
and diversity are correlated with the diameter of granu-
lar skin glands in juvenile and adult O. pumilio (Sapo-
rito et al. 2010). It is not known whether the quantity of

Table 1  Lengths and staging Gosner stage Stage characteristics Total

characteristics of tadpoles length
examined in developmental (mm)
series of Oophaga pumilio
25 No gills or limb buds 10.0
25 No gills or limb buds 10.4
27 Hind limb buds >½ and <1 times as long as wide 14.9
32 Hind limb buds “mitten” shaped 18.1
33 Hind limb buds with 3 early digits 17.9
40 Subarticular tubercles and cloacal tail piece, no forelimbs 21.4
41 Forelimbs visible under skin 21.9

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222 Zoomorphology (2017) 136:219–224

Fig. 2  Microphotographic images (×20) of granular skin gland are visible. Gland morphogenesis complements this species’ ontoge-
development in Oophaga pumilio, which starts with clusters of netic alkaloid profile in which alkaloids were detected in tadpoles
epithelial cells in stage 32 and alveoli forming in stage 33 (green stage 34 and above, but not in stage 25. Scale bars measure 50 μm
arrows). By stages 40–41, glands containing granules (red arrows)

alkaloid that is consumed or provisioned can influence tadpoles of confamiliar Dendrobates auratus were pro-
the timing of differentiation, growth, or ultimate size ducing “secretory aggregate” in their glands as early as
of granular glands or vice versa (Saporito et  al. 2010; stage 38. The chemical identity of such granules as well
Stynoski et  al. 2014a). In the current study, granular as those seen in stage 40 and 41 O. pumilio tadpoles in
glands differentiated and were forming granules (presum- the current study are not likely to be a toxic or unpalata-
ably alkaloid-free) in the absence of alkaloid consump- ble chemical, as captive-reared dendrobatids at any stage
tion. Angel et  al. (2003) also found that captive-reared are well known to be neither toxic nor noxious (Saporito

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Zoomorphology (2017) 136:219–224 223

Fig. 3  Microphotographic image of granular skin glands in stage 40

Oophaga pumilio tadpole showing the presence of translucent gran-
ules

et al. 2012; Santos et al. 2016 and therein); perhaps, these

granules help to maintain exogenous alkaloids when
available. Adults of a dendrobatid frog that does not
sequester alkaloids, Mannophryne [=Colostethus] trini-
tatis, also exhibit granules in granular glands (Neuwirth
et al. 1979; Grant et al. 2006; Santos et al. 2016). Future
work using labeled alkaloids or other chemical imaging
methods would confirm the ability of O. pumilio tadpoles
to store alkaloids in early glands and/or in other tissues.
The differentiation of granular glands in the late pre-
metamorphic stages coincides temporally across anurans,
including O. pumilio, with no obvious trends of hetero-
chrony. In amphibian taxa basal to dendrobatids, such as
the bufonid toads, larvae generate granular glands and
synthesise chemical defences concurrently in pre-met-
amorphic development (Chammas et  al. 2015). In later
life stages, those granular glands are mostly used for stor-
age of chemicals with minimal biosynthesis (Angel et al.
2003). The ability seen in poison frogs to sequester and
Fig. 4  Stereophotographic images (×3) of the mouthparts of
store lipid-soluble diet-based alkaloids has evolved inde- Oophaga pumilio tadpoles at stages 25, 33, and 41. Development
pendently in at least five amphibian families (Saporito includes elaboration of oral disc and its papillae, expansion and fur-
et  al. 2012; Santos et  al. 2016), perhaps from co-opted ther serration of the upper jaw sheath, and the addition of a posterior
glands that historically were biosynthesising other chemi- larval keratodont row
cal defences, such as peptides, steroids, and biogenic
amines, which in turn are thought to have evolved from Acknowledgements  The authors thank Kim Hoke for equipment
regulatory mucous glands (Neuwirth et  al. 1979; Daly use and Alexandre-Benoit Roland for animal care. JLS is supported
by an American Association for University Women American Post-
et  al. 1987; Toledo and Jared 1995). Ontogenetic func- doctoral Fellowship, and LAO is supported by a Bauer Fellowship
tional and transcriptomic analyses that explain uptake from Harvard University, the L’Oreal Women in Science Fellowship,
and storage in a comparative context would illuminate the the William F. Milton fund from Harvard Medical School, a Konishi
repeated evolution of alkaloid sequestration in tadpole Research Grant from the International Society for Neuroethology, and
the National Science Foundation (IOS-1528866).
and frog skin.

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224 Zoomorphology (2017) 136:219–224

Author contribution  JLS processed samples and wrote the man- Phylogenetic systematics of dart-poison frogs and their relatives
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Conflict of interest  The authors declare that they have no conflict
in eggs of the rough-skin newt, Taricha granulosa, are correlated
of interest.
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