Skin of the Red Eye Tree Frog Agalychnis Callidryas (Hylidae, Phyllomedusinae) Contains Lipid Glands of the Type Described in the Genus Phyllomedusa
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COMMENTARY THE ANATOMICAL RECORD 300:503–506 (2017) Skin of the Red Eye Tree Frog Agalychnis Callidryas (Hylidae, Phyllomedusinae) Contains Lipid Glands of the Type Described in the Genus Phyllomedusa ELISA ROTA,1 GIANFRANCO TANTERI,2 GILBERTO MONTORI,2 2 2 3 FILIPPO GIACHI, GIOVANNI DELFINO, * AND DAVID M. SEVER 1Department of Life Sciences and Biotechnologies, University of Ferrara, Ferrara, Italy 2Department of Biology, University of Florence, Florence, Italy 3Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana ABSTRACT Several anuran species of the genus Phyllomedusa are known to pos- sess specialized cutaneous glands producing lipids and exhibit a peculiar wiping behavior. This behavior is a stereotyped repertory of fore and hind limb movements distributing hydrophobic molecules onto the body surface and reducing evaporative water loss. No reports are presently available on the occurrence of lipid glands in other phyllomedusine genera, and data on the structure of the secretory units specialized for the production of cutaneous lipids are still unclear. The present report is aimed to answer both questions: it describes lipid glands of the Phyllomedusa type in Agalychnis callidryas and provides light and transmission electron microscope evidence of the syncytial structure of their secretory units, a typical feature of serous glands in anuran skin. This morphological trait supports the hypothesis that lipid glands are a specialized subset of the anuran serous glands, and underlines their flexible role in the skin adap- tion to sub-aerial environments. Anat Rec, 300:503–506, 2017. VC 2016 Wiley Periodicals, Inc. Key words: anuran skin; lipid glands; histology; ultrastructure; Hylidae Dorsal skin in several tree frog species regulates evapo- et al., 2013). Observed with the transmission electron rative water loss through surface lipids (Withers et al., microscope (TEM), the cutaneous lipids of monkey 1984) produced by cutaneous glands pertaining to the frogs appear as discrete vesicles (Blaylock et al., 1976) or ordinary mucous type (Lillywhite et al., 1997) or serous granules (Lacombe et al., 2000), consisting of light and type (Barbeau and Lillywhite, 2005), or by macroglands in the head skin (Warburg et al., 2000). The ordinary mucous and serous cutaneous glands synthesize hydrophobic mol- ecules along with their constitutive products, respectively *Correspondence to: Giovanni Delfino, Dipartimento di Biolo- gia, via La Pira 4 Universita di Firenze, Firenze 50121, Italy. proteoglycans or proteins. Hylid frogs of the type-genus Fax: 139-055-2756318 E-mail: [email protected] Phyllomedusa (the monkey tree frogs) possess specialized Received 19 April 2016; Revised 21 June 2016; Accepted lipid glands, which produce a pale secretory material 8 July 2016. (Antoniazzi et al., 2013) difficult to detect in ordinary light DOI 10.1002/ar.23502 microscope (LM) preparations (Lacombe et al., 2000), but Published online 14 October 2016 in Wiley Online Library positive to Sudan stains (Blaylock et al., 1976; Antoniazzi (wileyonlinelibrary.com). VC 2016 WILEY PERIODICALS, INC. 504 ROTA ET AL. moderately opaque rod-like subunits (Delfino et al., LIPID GLANDS DEVELOP IN TADPOLES 1998a,b; Nosi et al., 2002; Sevinc et al., 2005). Further- OF AGALYCHNIS CALLIDRYAS BEFORE more, ultrastructural studies suggested that lipid glands THE METAMORPHIC CLIMAX in Phyllomedusa represent a specialized type of the serous line, based on the syncytial structure of their secretory Lipid glands were detected from four limbed tadpoles units (Delfino et al., 1998a,b; Nosi et al., 2002; Sevinc (stage 42), (Fig. 1A), and already exhibited the distinc- et al., 2005). The Phyllomedusinae distribute lipids on tive mature traits found in adult frogs (Fig. 1B). The the body surface through a stereotyped wiping behavior early onset of lipid production in the skin during the (Blaylock et al., 1976) that occurs with a simpler repertory aquatic life stages was in agreement with previous in Hylinae (Barbeau and Lillywhite, 2005), and also investigations (Delfino et al., 1998a; Lacombe et al., evolved independently in arboreal frogs in the family Rha- 2000). The most remarkable feature of mature lipid glands was a lumen located in the upper half of the cophoridae (Lillywhite et al., 1997). Extending their inves- gland, and about half the width of the diameter of the tigation to other phyllomedusine genera, Blaylock et al. secretory unit (Fig. 1A,B). This cavity was integrally (1976) found scanty and small lipid glands in Pachyme- enclosed in the gland neck (Delfino et al., 1998a,b; Nosi dusa dacnicolor, whereas Agalychnis annae apparently et al., 2002): its lateral walls and floor were lined by lacked secretory units of this type. undifferentiated cells, intercalated between the duct and During a TEM investigation on the ontogenesis of the secretory unit (Fig. 1A–D). Cells of the floor were flat epidermis in the red eye tree frog Agalychnis callidryas and interdigitating at their tips without intervening des- (Giachi et al., 2011) cutaneous glands were occasionally mosomes (Fig. 1E). Due to the lack of intercellular junc- observed that resembled lipid glands of the phyllomedu- tions, local gaps were sometimes formed, and the sine type. A. callidryas does not exhibit wiping behavior, secretory product was released into the lumen (Nosi and has only moderately waterproof skin (Withers et al., et al., 2002; and Fig. 1A). Nematodes penetrating the 1984). These findings were confirmed in later obser- gland duct from the external environs (Sevinc et al., vations, and inspired the present report that extends 2005) can reach the syncytium through these gaps. Close the taxonomic distribution of lipid glands in tree frogs laminar formations, possibly resulting from layers of lip- through analytical comparisons between patterns de- id residues from secretory release, were visible in the tected in Agalychnis and Phyllomedusa. The analysis intercalated tract lumen (Fig. 1D). However, no lipid involved morpho-functional markers (Delfino et al., deposits were found in the neck lumen, because they 1998a,b; Nosi et al., 2002), including syncytial secretory were stored in the cytoplasm of the secretory unit (Fig. units and intracytoplasmic product storage. 1C,D, and Delfino et al., 1998a,b; Nosi et al., 2002; Sev- inc et al., 2005; Antoniazzi et al., 2013). These findings of intracytoplasmic secretory storage are different from ANIMALS AND METHODS previous reports: Blaylock et al., (1976) and Lacombe Agalychnis callidryas is an abundant species recorded et al. (2000) claimed that lipid deposits are stored in the as Least Concern in the IUCN red list (http://www.iucn- gland lumen, namely in a hollow compartment. How- redlist.org). A total of 6 specimens were investigated, ever, Figures 8A, B (Blaylock et al., 1976) and 6B including 2 adults and 4 tadpoles ranging between (Lacombe et al., 2000) of their articles provide evidence stages 28 and 44 of Gosner (1960). Animals were collect- of intracytoplasmic lipid storage in solid secretory units. ed and processed for preliminary steps of TEM investi- The lipid deposits exhibited variable features among gation in Gamboa (Panama), June-August 2008, by Dr. which droplets of low to intermediate opacity (Fig. 1C, Giachi in the research field facilities of the Smithsonian D), and peculiar aggregations of subunits with alternat- ing lighter and denser zones (Fig. 1F), which indicate an Tropical Research Institute, according to the policy for uneven distribution of lipids before post-fixation with wild animal care of that institution. As additional atten- OsO (Blaylock et al., 1976). The secretory syncytium tion, tadpoles were reared from embryos hanging on 4 was adjacent to the laminar cells of the lumen floor (Fig. muddy ponds unsuitable for larval development, and 1D), but it was separated from the peripheral sheath of specimens not employed in investigation were released contractile (myoepithelial) cells by an obvious intercellu- into moist environments after metamorphosis (Giachi lar space (Fig. 1G,H). This interstice contained slender et al., 2011). Dorsal skin strips were removed and and short outgrowths from secretory syncytium and treated with the aldehyde solution of Karnovsky (1965) myoepithelial cells (Fig. 1H), along with larger and lon- and, once transported to the Laboratory of Comparative ger cytoplasmic processes with an electron-transparent Anatomy of the Department of Evolutionary Biology, ground substance, holding a cytoskeleton of parallel University of Florence (currently BIO UNIFI), they microtubules and scattered vesicles with dense content underwent further steps of preparation (OsO4 fixation, (Fig. 1G). These processes were neurites (Nosi et al., dehydration, EPON embedding and polymerization). 2002) containing dense-cored synaptic vesicles in their Semithin and ultrathin sections, for LM and respectively endings (Delfino et al., 1998b). TEM observations, were obtained with LKB ultramicro- In tadpoles, lipid glands exhibited large peripheral tomes equipped with diamond knives, and were stained areas of the secretory syncytium rich in smooth and rough through routine procedures. Digital LM images were col- endoplasmic reticulum (Fig. 1I), whereas in adult glands lected with a LEITZ DMRB microscope equipped with a the peripheral cytoplasm contained scanty biosynthesis NIKON COOLPIX 4500 camera. Analogic TEM images organelles (Fig. 1H,L) in a dense ground substance, with- were collected