A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation.

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

Cultivating Positive Youth Development, Critical Consciousness, and Authentic Care in Urban Environmental Education.

This paper addresses the issue of how to provide affordances for youth development in the context of environmental stewardship in cities. Urban environmental education encompasses place-based and action-oriented stewardship practices, including community gardening and vegetable production, often with the dual goals of developing youth and community assets. Yet in-depth understanding of how these goals are achieved is lacking. Using narrative inquiry, we explored participant experiences in a multi-year agriculture internship program conducted by the food justice organization East New York Farms! (ENYF) in Brooklyn, NY. Emerging from our conversations with youth were five themes defining their intern experience: ENYF as somewhere to belong, to be pushed, to grapple with complexity, to practice leadership, and to become yourself. We propose a theory of change that emphasizes politicized notions of caring as a foundation for cultivating developmental assets, including competence, contribution, and critical consciousness, among youth who participate in ENYF programs multiple years. This paper extends the literature on socio-environmental affordances to encompass urban environmental education programs, which incorporate physical and social features that act as affordances. Further, this paper describes a feedback loop in which youth afforded opportunities to develop assets through contributing to their community in turn create affordances for additional youth and adults.

A review of Northern Peruvian glassfrogs (Centrolenidae), with the description of four new remarkable species.

Peru is well known for amphibian diversity and endemism, yet there have been relatively few field studies of glassfrog (Centrolenidae) diversity in this country. Research in Colombia and Ecuador indicates that centrolenid diversity is higher in the northern Andes. However, part of this trend appears to be due to sampling effort. We conducted fieldwork throughout northern Peru, and based on phylogenetic analysis of DNA sequences, combined with bioacoustic and morphological analyses of new and available material we now recognize 33 species from the country (versus 30 species prior to this work). Field surveys led to the discovery of four remarkable species: Centrolene charapita new species is a large, ornamented glassfrog that appears to be sister to Ce. geckoideum; Chimerella corleone new species represents the second-known member of the genus Chimerella; Cochranella guayasamini new species is the second-known member of the genus with humeral spines; and Hyalinobatrachium anachoretus new species occurs in the cloud forest of the east-Andean versant in Peru. In addition to the new species described here, we provide new country records, new localities including range extensions of up to 875 km, information on diagnostic characters and phylogenetic relationships, call and larval descriptions, and observations on natural history for several Peruvian centrolenids. Our results also revealed several taxonomic problems concerning species of the genus Rulyrana, and we conclude that R. croceopodes and R. tangarana are junior synonyms of R. saxiscandens. By implication of our phylogenetic analyses, we recognize the following new combinations: Espadarana audax new combination, Espadarana durrelorum new combination, and Espadarana fernandoi new combination. 

Glassfrog embryos hatch early after parental desertion.

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.