Growth and uptake of mineral by embryos of the direct-developing frog Eleutherodactylus coqui

Embryos of the direct-developing frog Eleutherodactylus coqui take up small quantities of yolk and yolk mineral early in incubation but increase their uptake of yolk reserves at later stages of development. Growth and accumulation of calcium and magnesium by embryos also occur slowly at first and at a higher rate later. Accumulation of calcium and magnesium by embryos is largely a function of variation in size of embryos, but uptake of phosphorus is unrelated to size. Althrough patterns of growth and uptake of mineral by embryonic coquis resemble those for embryos of oviparous amniotes, embryonic coquis do not deplete the yolk of its nutrients to the same degree. Thus, residual yolk of coqui hatchlings contains a high percentage of the nutrient reserves originally present in the egg. This difference between embryonic coquis and embryos of oviparous amniotes may indicate that transfer of nutrients from yolk to embryo becomes limiting during the grwoth phase. Alternatively, some aspects of the neurologic system are so poorly developed at hatching that coqui may not be able to find prey effectively. A large nutrient reserve could sustain hatchling while the neurologic system continues to mature.     

Development of the retinotectal system in the direct-developing frog Eleutherodactylus coqui in comparison with other anurans

Background

Associations between animal species require that at least one of the species recognizes its partner. Parabioses are associations of two ant species which co-inhabit the same nest. Ants usually possess an elaborate nestmate recognition system, which is based on cuticular hydrocarbons and allows them to distinguish nestmates from non-nestmates through quantitative or qualitative differences in the hydrocarbon composition. Hence, living in a parabiotic association probably necessitates changes of the nestmate recognition system in both species, since heterospecific ants have to be accepted as nestmates.

Results

In the present study we report highly unusual cuticular profiles in the parabiotic species Crematogaster modiglianii and Camponotus rufifemur from the tropical rainforest of Borneo. The cuticle of both species is covered by a set of steroids, which are highly unusual surface compounds. They also occur in the Dufour gland of Crematogaster modiglianii in high quantities. The composition of these steroids differed between colonies but was highly similar among the two species of a parabiotic nest. In contrast, hydrocarbon composition of Cr. modiglianii and Ca. rufifemur differed strongly and only overlapped in three regularly occurring and three trace compounds. The hydrocarbon profile of Camponotus rufifemur consisted almost exclusively of methyl-branched alkenes of unusually high chain lengths (up to C₄₉). This species occurred in two sympatric, chemically distinct varieties with almost no hydrocarbons in common. Cr. modiglianii discriminated between these two varieties. It only tolerated workers of the Ca. rufifemur variety it was associated with, but attacked the respective others. However, Cr. modiglianii did not distinguish its own Ca. rufifemur partner from allocolonial Ca. rufifemur workers of the same variety.

Conclusion

We conclude that there is a mutual substance transfer between Cr. modiglianii and Ca. rufifemur. Ca. rufifemur actively or passively acquires cuticular steroids from its Cr. modiglianii partner, while the latter acquires at least two cuticular hydrocarbons from Ca. rufifemur. The cuticular substances of both species are highly unusual regarding both substance classes and chain lengths, which may cause the apparent inability of Cr. modiglianii to discriminate Ca. rufifemur nestmates from allocolonial Ca. rufifemur workers of the same chemical variety.     

Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.

Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of microsatellite loci from the coqui frog, Eleutherodactylus coqui

Thirteen microsatellite loci were isolated from the coqui frog (Eleutherodactylus coqui) and optimized for future research. The loci were screened across 37 individuals from two Puerto Rican populations. Loci were variable with the number of alleles per locus ranging from three to 38. Polymorphic information content ranged from 0.453 to 0.963 and observed heterozygosity for each population ranged from 0.320 to 0.920.     

Cranial neural crest-cell migration in the direct-developing frog, Eleutherodactylus coqui: molecular heterogeneity within and among migratory streams

Direct development is a specialized reproductive mode that has evolved repeatedly in many different lineages of amphibians, especially anurans. A fully formed, albeit miniature adult hatches directly from the egg; there is no free-living larva. In many groups, the evolution of direct development has had profound consequences for cranial development and morphology, including many components that are derived from the embryonic neural crest. Yet, the developmental bases of these effects remain poorly known. In order to more fully characterize these changes, we used three molecular markers to analyze cranial neural crest-cell emergence and migration in the direct-developing frog, Eleutherodactylus coqui: HNK-1 immunoreactivity, Dlx protein expression, and cholinesterase activity. Our study validates and extends earlier results showing that the comprehensive changes in embryonic cranial patterning, differentiation, and developmental timing that are associated with direct development in Eleutherodactylus have not affected gross features of cranial neural crest biology: the relative timing of crest emergence and the number, configuration and identity of the principal migratory streams closely resemble those seen in metamorphic anurans. The three markers are variably expressed within and among neural crest-cell populations. This variation suggests that determination of cranial neural crest-cells may already have begun at or soon after the onset of migration, when the cells emerge from the neural tube. It is not known how or even if this variation correlates with differential cell lineage or fate. Finally, although HNK-1 expression is widely used to study neural crest migration in teleost fishes and amniotes, E. coqui is the only amphibian known in which it effectively labels migrating neural crest-cells. There are not enough comparative data to determine whether this feature is functionally associated with direct development or is instead unrelated to reproductive mode.     

Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals.     

Fertilization and aqueous development of the puerto rican terrestrial-breeding frog,Eleutherodactylus coqui

The Puerto Rican tree frog, Eleutherodactylus coqui, has internal fertilization and direct development on land. In light of these reproductive adaptations, the events of fertilization and early development were studied. Cytological examination of just-fertilized eggs showed that sperm entry is restricted to about 10% of the surface of these large, yolky eggs, and all nuclear events of the first cell cycle occur near the animal pole. Although the oocytes have cortical granules, a number of polyspermic fertilizations were found. One clutch consisted of eggs with a high frequency of polyspermy and of normal development. This raises the possibility that normal development can occur despite multiple sperm entry, a situation not found in other anuran amphibians. With respect to saline requirements, the sperm and the embryo are similar to those in amphibians with external fertilization and aqueous development. Sperm motility was high in low-tonicity conditions, and the normally terrestrial embryo could develop completely from a fertilized egg to a froglet in a low-tonicity aqueous solution.     

Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui

World-wide reports of amphibian population declines have led to increased interest in the reproductive biology of anurans. As a model system, here we present evidence for the effective cryoprotection of sperm from the Puerto Rican frog, Eleutherodactylus coqui, using mixtures of fetal bovine serum (FBS) and dimethylsulfoxide (Me(2)SO), glycerol or sucrose extenders. Using a fluorescent dye exclusion assay, we found that 53.9 and 50.4% of all sperm with intact membranes prior to freezing maintained membrane integrity after rapid freezing and thawing when protected with either a FBS/glycerol or FBS/sucrose solution, respectively. The methods reported here may be useful for similar work with many of the more than 700 other species in this genus.     

Mesoderm formation in Eleutherodactylus coqui: body patterning in a frog with a large egg.

The direct developing frog, Eleutherodactylus coqui, develops from a large egg (diameter 3.5 mm). To investigate the effect of egg size on germ-layer formation, we studied mesoderm formation in E. coqui and compared it to that of Xenopus laevis (diameter 1.3 mm). First, we identified the position of prospective mesoderm in the 16-cell E. coqui embryo by cell-lineage tracing. Although the animal blastomeres are small, they form most of the blastocoel roof and make extensive contributions to some mesodermal tissues. Second, we performed recombinant analysis with X. laevis animal caps to define the distribution of mesoderm-inducing activity. Mesoderm-inducing activity in E. coqui was restricted around the marginal zone with strong activity in the superficial cells. Neither the vegetal pole nor the blastocoel floor had activity, although these same regions from X. laevis induced mesoderm. Third, we cloned Ecbra, a homologue of Xbra, an early mesoderm marker in X. laevis. Ecbra was expressed in the marginal ring close to the surface, similar to X. laevis, but E. coqui had weaker expression on the dorsal side. Our results suggest that mesoderm formation is shifted more animally and superficially in E. coqui compared to X. laevis.     

Molecular anatomy of the developing limb in the coquí frog,Eleutherodactylus coqui

The vertebrate limb demonstrates remarkable similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this morphological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size‐reduced embryos of the Puerto Rican coquí frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life‐history strategy marked by rapid progression from egg to adult and absence of a free‐living, aquatic larva. Nonetheless, coquí exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We investigated the extent to which coquí limb bud development conforms to the model of limb development derived from amniote studies. Toward this end, we characterized dynamic patterns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are essentially unchanged compared to amniote species. For example, we identified an EcFgf8‐expression domain within the apical ectodermal ridge (AER). This expression pattern defines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coquí embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate altered domains of expression, which imply a potential shift in gene function between coquí frogs and amniote model systems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expression pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expression of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of ancient limb‐patterning molecules, which likely mediate identical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic control of a conserved morphological pattern across evolutionary time.     

Characterization of the nutritional endoderm in the direct developing frog Eleutherodactylus coqui

Unlike Xenopus laevis, Eleutherodactylus coqui develops without a tadpole. The yolk-rich vegetal region of the embryo forms a transient nutritive tissue, the nutritional endoderm (NE). The definitive endoderm (DE) in E. coqui comes from cells closer to the animal pole in contrast to its vegetal origin in X. laevis. RNA important for initiating the endoderm specification network is absent in presumptive NE cells, raising the question whether signaling occurs in them. We explored the nature of NE and asked how differences between NE and DE cells arise. We identified differences between NE and DE that first become evident at gastrula, when NE cells become multinucleated. Nuclear β-catenin, an essential cofactor of sox 17, important for endoderm formation in X. laevis, is present in NE and DE at gastrula but remains in NE long after it is not seen in DE. We cloned E. coqui homologs of TGFβs activin b and derriere and provide evidence for their maternal expression. We also detected activin b and derriere RNAs in NE at gastrula and show that NE possesses some mesoderm-inducing activity, but it is delayed with respect to DE. Our findings indicate that altered development of NE begins at gastrula. RNAs important for mesendoderm induction and some mesoderm-inducing activity are present in NE.     

Taste organ growth and development in the direct developing frog Eleutherodactylus coqui (Lissamphibia: Eleutherodactylidae)

Previous research on amphibian taste organs concerned amphibians with a biphasic life history, that is, with larval period and metamorphosis. Direct developing frog species, such as Eleutherodactylus coqui, undergo a cryptic metamorphosis before hatching, and many larval‐specific features are vestigial or have been lost entirely from their ontogeny. Taste buds are present in larval stages of biphasically developing anurans and are replaced by taste discs during metamorphosis. One goal of this study was to characterize the ontogeny of taste buds and/or discs in E. coqui. The other goal was to examine correlations between body size and taste organ density and size in different regions of oral epithelium. The research reveals the presence of only one type of taste organ, characteristic of metamorphs of biphasic amphibians, namely taste disc. In addition, taste disc density and the area of the taste disc sensory zone change dramatically during growth.     

Arginine vasotocin activates advertisement calling and movement in the territorial Puerto Rican frog, Eleutherodactylus coqui

Arginine vasotocin (AVT) is a neuropeptide that modulates social behavior in amphibians and activates calling in frogs. The Puerto Rican coqui frog, Eleutherodactylus coqui, is a terrestrial anuran that exhibits complex social behaviors, including territoriality and paternal care. Males have a distinctive social hierarchy and can be any of the following: satellite (non-calling), territorial (calling), or paternal (guards and broods embryos). Field experiments were conducted to determine the effects of AVT on satellite behavior and the male social hierarchy of E. coqui. Satellite males were captured within the territory held by a resident male, given injections (i.p.) of AVT or saline (control) and placed back in their original location. To determine if AVT affects all males, not merely satellite males, territorial (calling) males were injected with AVT. Significantly more satellite males commenced advertisement calling following AVT injections than did control males injected with saline. AVT-activated satellites did not challenge the resident territorial male for possession of the territory but instead moved into a new area before commencing to call. In fact, AVT-activated satellite males were significantly more likely to move into a new territory following AVT injections than AVT-injected territorial males. The effect of AVT was short lived, lasting only one night in all but two cases. It is concluded that AVT stimulates advertisement calling and AVT-activated males displayed territorial characteristics of E. coqui.     

Loss of ectodermal competence for lateral line placode formation in the direct developing frog Eleutherodactylus coqui.

In the direct-developing frog Eleutherodactylus coqui neuromasts and ganglia of the lateral line system never develop. We show here that this absence of the lateral line system, which is evolutionarily derived in anurans, is due to very early changes in development. Ectodermal thickenings, which are typical of lateral line placodes, and from which neuromasts and ganglion cells of the lateral line originate, never form in E. coqui, although other neurogenic placodes are present. Moreover, although NeuroD is expressed in the lateral line placodes of Xenopus laevis, corresponding expression sites are lacking in E. coqui. Heterospecific transplantation experiments show that axolotl ectoderm can be induced to form lateral line placodes after transplantation to E. coqui hosts but that E. coqui ectoderm does not form lateral line placodes on axolotl hosts. This suggests that the loss of the lateral line system in E. coqui is due to the specific loss of ectodermal competence to form lateral line placodes in response to inductive signals. Our results (1) indicate that the competence for lateral line placode formation is distinct and dissociable from the competence to form other neurogenic placodes and (2) support the idea that the lateral line system acts as a module in development and evolution.     

Opercular development and ontogenetic re-organization in a direct-developing frog

The direct-developing frog, Eleutherodactylus coqui, has eliminated the tadpole stage from its ontogeny, and lacks many larval characters. We demonstrate that the dermal folds of E. coqui are homologous with the opercular folds of metamorphosing frogs. In both E. coqui and its metamorphic counterparts the opercular folds grow over the developing forelimb before perforating to free the entrapped limb. Opercular perforation in E. coqui occurs even in the absence of the forelimb but shows no signs of thyroid hormone dependence. The condensation of E. coqui development appears due to the excision of the extended larval period of developmental stasis. Analysis of opercular development, when viewed in conjunction with other developmental characters, suggests the ontogenetic period in the ancestral Eleutherodactylus life-history from which the tadpole was likely eliminated. Received: 7 October 1999 / Accepted: 17 January 2000