DEVELOPMENTAL DYNAMICS 238:1444–1454, 2009 SPECIAL ISSUE TECHNIQUES Embryogenesis and Laboratory Maintenance of the Foam-Nesting Tu´ ngara Frogs, Genus (Physalaemus ؍) Engystomops Andre´s Romero-Carvajal,1 Natalia Sa´ enz-Ponce,1 Michael Venegas-Ferrı´n,1 Diego Almeida-Reinoso,2 Chanjae Lee,3 Jennifer Bond,4 Michael J. Ryan,4 John B. Wallingford,3 and Eugenia M. del Pino1* The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of tu´ ngara frogs (genus Engystomops, also called Physalaemus). The frogs Engystomops pustulosus, Engystomops coloradorum, and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. Developmental Dynamics 238:1444–1454, 2009. © 2009 Wiley-Liss, Inc. Key words: gastrulation modes; somitogenesis; neural development; Colostethus machalilla; Engystomops coloradorum; Engystomops randi; Engystomops pustulosus; Gastrotheca riobambae Accepted 10 March 2009 INTRODUCTION ing the rainy season; the male attracts ing tadpoles of other frogs (Ryan, the female with a characteristic call, 1985). Moreover, the hardened sur- Many frogs deposit their eggs in the and amplexus takes place. During am- face of the nest may reflect solar radi- water; other frogs, however, have evolved adaptations that diminish or plexus, the male beats the egg-jelly ation, decreasing heat absorption, and eliminate the aquatic requirement for with its legs and feet. This action pro- preventing internal desiccation. At reproduction (Duellman and Trueb, duces a floating mass of white foam, in hatching, the tadpoles fall into the wa- 1986). A deviation of the aquatic re- which numerous eggs are embedded ter (Davidson and Hough, 1969; Ryan, productive mode occurs in the tu´ ngara (Davidson and Hough, 1969; Ryan and 1985). Engystomops pustulosus is ac- frogs, genus Engystomops (ϭ Physa- Rand, 2003). In this way, E. pustulo- tive at night and displays a complex laemus), Leiuperidae, and consists of sus eggs are placed over, and develop reproductive behavior. The ecology, the formation of foam-nests that float above, water. The strategy of placing sexual selection, and communication over temporal pools. In Engystomops foam-nests over temporal pools pro- strategies of this frog have been exten- pustulosus, reproduction occurs dur- tects embryos from predators, includ- sively studied (reviewed in Ryan, Additional Supporting Information may be found in the online version of this article. 1Laboratorio de Biologı´a del Desarrollo, Escuela de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica del Ecuador, Quito, Ecuador 2Museo de Zoologı´a, Seccio´n de Herpetologı´a, Escuela de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica del Ecuador, Quito, Ecuador 3Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology University of Texas, Austin, Texas 4Section of Integrative Biology, University of Texas, Austin, Texas Grant sponsor: Pontificia Universidad Cato´lica del Ecuador; Grant sponsor: The Academy for the Developing World (TWAS); Grant number: 07-017 LDC/BIO/LA-UNESCO FR 3240144821; Grant sponsor: NIH/NIGMS. *Correspondence to: Eugenia M. del Pino, Pontificia Universidad Cato´ lica del Ecuador, Escuela de Ciencias Biolo´ gicas, Apartado 17-01-2184, Avenida 12 de Octubre 1076 y Roca, Quito, Ecuador. E-mail: [email protected] DOI 10.1002/dvdy.21952 Published online 20 April 2009 in Wiley InterScience (www.interscience.wiley.com). © 2009 Wiley-Liss, Inc. EMBRYOS OF FOAM-NESTING FROGS 1445 1985; Ryan and Rand, 2003; Ron et Pino et al., 2007). Furthermore, as in RESULTS AND DISCUSSION al., 2006). X. laevis, embryonic development oc- Frogs of the genus Engystomops in- curs rapidly in comparison with the Engystomops Developmental habit the forest floor in lowland re- slow-developing embryos of the den- Stages gions from Central Mexico to the Am- drobatid frog Colostethus (ϭ Epipe- Development of E. pustulosus, E. colo- azon basin, and the lowlands of W dobates) machalilla (Grant et al., radorum, and E. randi embryos in the Ecuador and NW Peru (reviewed by 2006), and of the marsupial frog Gas- Ron et al., 2006). The widely studied, foam-nest was divided into 23 stages trotheca riobambae (del Pino et al., (st). Development in the foam-nest Engystomops pustulosus, occurs at the 2007). Tail bud stage embryos of E. Smithsonian Tropical Research Insti- lasted 3 days in all of these frogs. At coloradorum and E. randi differ from hatching, the foam-nest became some- tute in Panama´, among other loca- X. laevis embryos in their general tions (Davidson and Hough, 1969; what liquid and the tadpoles moved shape, in the mode of somitogenesis, into the water. The criteria used to Ryan, 1985; Ron et al., 2006). Other and size of cranial neural crest cell congenerics that have been analyzed define the developmental stages are streams (del Pino et al., 2007). are Engystomops coloradorum, and E. given in Table 1. Developmental Analysis of Lim1 (Lhx1) gene ex- randi, which inhabit the western low- stages from fertilization to the early pression at the protein level in gastru- lands of Ecuador (Ron et al., 2006). neurula were defined according to the lae of E. coloradorum and E. randi Mitochondrial DNA analysis of these X. laevis table of development (st allowed detection of the organizer, three species revealed a close relation- 1–14; Nieuwkoop and Faber, 1994). ship. Accordingly, these frogs have and of the dorsal mesoderm (del Pino Thereafter, the generalized table of been placed in the Engystomops pus- et al., 2007; Venegas et al., 2009). In frog development (Gosner, 1960) was tulosus species group (Ron et al., embryos of these frogs, elongation of used to stage embryos, as the embry- onic morphology of Engystomops devi- 2006). In this study, we describe the the notochord overlaps with involu- ated from that of X. laevis embryos early development of these Engysto- tion at the blastopore lip, as in X. lae- (Table 1) The external views of em- mops species, methods for the han- vis embryos (del Pino et al., 2007). In bryos are provided to complement the dling of eggs and embryos, and frog addition, in the mid gastrula, the pre- description of developmental stages laboratory maintenance. chordal plate and notochord were si- (Figs. 1A–P, 2A–G). The free-living In the search for an anuran amphib- multaneously recognized by the ex- tadpoles were not studied. ian with synchronous oogenesis, Da- pression of the protein Lim1, a The foam-nests of E. pustulosus, E. vidson and Hough (1969) determined pattern equivalent with that of Xlim1 coloradorum, and E. randi measured that E. pustulosus was appropriate for mRNA expression in X. laevis em- about 5 cm in diameter and were 4–5 developmental work. The environ- bryos (Taira et al., 1992; 1994a,b; cm high. The white eggs and embryos mental changes of wet and dry sea- Venegas et al., 2009). In contrast, in of these frogs were effectively camou- sons were reproduced in the labora- embryos of the dendrobatid frog C. flaged in the white foam of the nest. tory to analyze the modifications of machalilla, only the prechordal plate Each nest contained 130 Ϯ 60 eggs of oogenesis. Oogenesis changes from was recognized during gastrulation. 1.3-mm diameter in E. coloradorum synchronous during the dry season to Elongation of the notochord occurred (21 foam-nests with a total of 2,072 asynchronous in the wet season. Dur- after blastopore closure in this frog eggs); and 110 Ϯ 69 eggs of 1.1-mm ing the dry season, many oocytes (Venegas et al., 2009). This uncou- diameter in E. randi (17 foam-nests reach their full growth (stage 6) and pling of developmental events associ- with a total of 1,952 eggs). The foam- remain in the ovary until reproduction ates with the modular nature of frog nests of E. pustulosus contain an av- is triggered by the first rains. Besides gastrulation (Ewald et al., 2004; Moya erage of 238 Ϯ 97 eggs, based on 68 these large oocytes, only previtello- et al., 2007; del Pino et al., 2007; Vene- foam-nests collected in the field at the genic oocytes were detected in the gas et al., 2009). Barro Colorado Island, Panama´ ovary by the end of the dry season. In In this study, we immunostained (Ryan, 1985). The means and stan- contrast, during the wet season, the Engystomops embryos with several dard deviations are given. full range of oocyte sizes occurs in the antibodies, to detect the neural tube, ovary. After the first mating of the cranial neural crest cells, somites, rainy season, ovarian oocytes grow, Cleavage and the Blastula pronephros, and performed in situ hy- producing oocytes of all sizes. There- (st 1–9) after, in preparation for the impend- bridization to analyze the expression ing mating, the largest oocytes com- of the RetinoBlastoma Binding Pro- External views of cleavage stage em- plete their growth. This adaptation tein 6 (RBBP6) during development. A bryos are shown in Figure 1A–E, and facilitates repeated mating during the particular advantage for developmen- of the blastula in Figure 1F,G. In the wet season. Maternal transcripts are tal studies is the brief life-cycle of three species analyzed, eggs had a retained in E. pustulosus embryos to these frogs, which can be as short as 4 uniform white appearance.