Zoology 113 (2010) 175–183 Contents lists available at ScienceDirect Zoology journal homepage: www.elsevier.de/zool Skeletal muscle–melanocyte association during tadpole tail resorption in a tropical frog, Clinotarsus curtipes Jerdon (Anura, Ranoidea) Lekha Divya a,1, Reston S. Beyo a,1, Parameswaran Sreejith a,1, Mohammad A. Akbarsha b,1, Oommen V. Oommen a,∗,1 a Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram 695581, India b School of Life Sciences, Bharathidasan University, Tiruchirapally 620024, India article info abstract Article history: We tested the hypothesis that melanin has a role as a molecule within the thyroid-mediated cascade. Received 17 June 2009 Light microscopic and ultrastructural changes in the skeletal muscle during tail resorption in tadpoles of Received in revised form 30 October 2009 the tropical frog Clinotarsus curtipes Jerdon (Anura: Ranoidea) were observed. Light microscopic analysis Accepted 6 November 2009 at metamorphic stage XVIII showed a melanized epidermis. A gradual migration of melanocytes from the epidermis to the dermis and filopodia of melanocytes pervading the skeletal muscle preceded tail Keywords: resorption. The invasion of melanocytes into the muscle bundles coincided with the breakdown of the Clinotarsus curtipes muscle bundles into sarcolytes and the arrival of macrophages at this site. This would suggest that the Melanin Filopodia melanocyte–sarcolyte association signals the arrival of macrophages at these sites as metamorphosis Sarcolytes progressed. Melanophages, macrophages with melanin granules, were observed at the climax stage of Metamorphosis XXIII. The sarcolytes and the melanin granules were phagocytosed by macrophages so as to completely cleanse the exocytic muscle debris and the melanin granules. The presence of large melanomacrophage centers in the tadpole liver at metamorphic climax suggests that these phagocytic macrophages were further processed in the liver and, likely, in the spleen. It is proposed that melanin, a byzantine molecule, has a role in the cascade of events leading to tail resorption in anuran tadpoles. © 2010 Elsevier GmbH. All rights reserved. 1. Introduction Anurans have been the focal organisms for understanding amphibian metamorphosis, primarily due to the dramatic nature The amphibian metamorphosis from tadpole to frog is one of of their metamorphosis and the easy handling of anuran species in the most enigmatic post-embryonic transformations. It encom- research. However, among the anurans, metamorphosis has been passes most, if not all, of the intra- and extra-cellular processes reasonably well studied only in three species, Xenopus laevis (South that are involved in vertebrate embryogenesis and organogenesis African clawed frog), Rana catesbeiana (bull frog) and Rana pipiens (Shi, 2000). The changes that take place in the tissues and organs (Northern Leopard frog), all temperate species. Although other anu- of the tadpole, as it transforms into a frog, have made it a model rans like Bufo and Rana species have also been studied, there is no for studying the post-embryonic organ remodeling and develop- report thus far on tail resorption in any tropical anuran. ment for nearly a century (Shi, 2000). Three primary morphological Clinotarsus curtipes Jerdon (1854) is widely distributed in the changes occur during metamorphosis: (i) resorption or regression Western Ghats of India. Known as the ‘leaf-litter frog’, it is found of tissues, organs or organ systems that have a primary function in a number of tropical forest types (evergreen to semi-evergreen only in the larva, (ii) remodeling of larval organs or organ systems moist and also dry deciduous forests). The tadpoles of C. curtipes into their adult form, and (iii) de novo development of tissues in the are the largest of any Indian tadpole known so far, although there adult that are not required by the larva. These changes are more are reports by Fabrezi et al. (2009) on giant tadpoles of Pseudis marked in anuran species and less obvious in urodeles and caecil- platensis Gallardo, 1961 having a prolonged metamorphic period ians. This entire panorama of events leads to the development of and also of Pseudis paradoxa by Emerson (1988). C. curtipes tad- an adult organism capable of living or surviving in a habitat very poles, which attain a length of nearly 10 cm, have an extended different from the one in which the tadpoles live. larval period lasting for about 6 months (Valamparampil, 1994) that makes them suitable for the study of stages in the developmen- tal changes. The tadpoles grow during the early stages of their life ∗ history and undergo metamorphosis with complete tail regression Corresponding author. Tel.: +91 471 2308906; fax: +91 471 2418906. E-mail address: [email protected] (O.V. Oommen). before turning into adults. C. curtipes tadpoles are darkly colored 1 All authors contributed equally to the manuscript. with dense melanophores in the skin and elsewhere. The tadpoles 0944-2006/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2009.11.002 176 L. Divya et al. / Zoology 113 (2010) 175–183 Fig. 1. Representative critical stages in the life cycle of Clinotarsus curtipes based on external morphology and morphometric index (left panel, photographs; right panel, line drawings). (A) Stage XVIII tadpole, (B) stage XXI, (C) stage XXIII, and (D) metamorphosed froglet. Scale bar = 1.3 cm. retain their black coloration from the early feeding stage to the cli- retention of strong tail muscle fibers until late during the life his- max of the metamorphic stage. Subsequently, they develop into the tory (Hiragond et al., 2001). These, at the end of metamorphosis, typical adult ‘bicolored frog’. suddenly collapse, giving rise to tail resorption. The process should The familiar black or brown coloration of amphibian tadpoles involve much more than the conventional apoptotic machinery. is usually attributed to melanin. Most life forms produce melanins, Resorption of the tail in the context of amphibian metamorpho- which are dark (usually black) and complex, poorly characterized sis involves degeneration of not only the skeletal muscle but also pigments, that are synthesized enzymatically or auto-oxidatively of the vertebral column/notochord, nerve cord, blood vessels, con- from a variety of cyclic, heterocyclic, phenolic or other resonance- nective tissue, etc. (Weber, 1964; Kerr et al., 1974; Kinoshita et al., stabilized precursor molecules. Melanins make up a heterogeneous 1985). Unlike the other tissues, which can be reabsorbed through class of natural pigments that have a myriad of biological functions the processes of apoptosis and/or lysosome-mediated necrosis, the (Nosanchuk and Casadevall, 2003). The presence of various kinds melanocytes of the skin do not succumb to either of these cell of melanins in almost every taxon suggests an evolutionary impor- death processes. A correlation has been reported between melanin tance. Up until now, however, there is no agreement concerning content and melanomacrophagic component during hibernation the primary or basic function of melanogenesis and its product in Rana esculenta (Barni et al., 2002). It is generally believed that melanin, or even concerning its character as a primary or a side the melanocytes of the tail skin provide melanin pigment for the effect (Plonka and Grabacka, 2006). post-metamorphic keratinization of the skin to the rest of the body. Anuran tail resorption has been used by several investigators as Purrello et al. (2001) reported that liver pigment cells containing an endpoint index for several morphological, histological, biochem- melanin underwent programmed cell death faster in comparison ical and molecular studies. The tail is genetically pre-determined with cells with low pigment content in R. esculenta. Therefore, to be resorbed, requiring only sufficient levels of thyroid hormones melanin itself might be involved in some way in the tadpole meta- to initiate the process (Valamparampil and Oommen, 1997). All morphosis. tissues that comprise the tadpole tail are resorbed during meta- morphosis, including the epidermis, connective tissue, skeletal muscle, blood vessels, notochord, etc. Kerr et al. (1974), in their 2. Materials and methods classic description of amphibian metamorphosis, suggested that the tail resorption occurs through a special kind of apoptosis. The Tadpoles of C. curtipes (Rana curtipes Jerdon) of various stages present paper describes the stage-wise morphological events asso- were maintained in the laboratory in fiberglass aquaria with aer- ciated with tail resorption in the tropical anuran, C. curtipes. The ated, dechlorinated tap water at conditions of ambient temperature so-called ‘prolonged metamorphosis’ of this species requires the (29 ± 2 ◦C) and photoperiod (approximately 12L:12D) and fed on L. Divya et al. / Zoology 113 (2010) 175–183 177 Fig. 2. Tail sections of Clinotarsus curtipes, embedded in paraffin and stained with nile blue sulphate. (A) Stage XVIII: showing epidermis (e), dermis (d), skeletal muscle (sm) and melanocytes (m); (B) stage XXI; (C) stage XXIII (*indicates melanin in deeper locations); (D) semi-thin tail section at stage XXIII showing sarcolytes (sl), migrating melanocytes (mg), blood vessel (bv) and empty space (es) surrounded by melanocytes. Toluidine blue O stain. Scale bars = 40 ␮m. boiled spinach (Amaranthus sp.) ad libitum. The tadpoles were Tadpoles were anesthetized by placing them on ice and trans- assigned