The ability to isolate and perform nucleic acid analyses of individual cells is critical to studying the development of various cell types and structures. We present a novel biological sample preparation method developed for laser capture microdissection-assisted nucleic acid analysis of ultrathin cell/tissue sections. We used cells of the mitotic bed of the tadpole teeth of Lithobates sphenocephalus (Southern Leopard Frog). Cells from the mitotic beds at the base of the developing teeth series were isolated and embedded in the methacrylate resin, Technovit® 9100®. Intact cells of the mitotic beds were thin sectioned and examined by bright-field and transmission electron microscopy. The cytological and ultrastructural anatomy of the immature and progressively more mature tooth primordia appeared well preserved and intact. A developmental series of tooth primordia were isolated by laser capture microdissection (LCM). Processing of these cells for RNA showed that intact RNA could be isolated. The study demonstrates that Technovit® 9100® can be used as an embedding medium for extremely small tissues and from individual cells, a prerequisite step to LCM and nucleic acid analyses. A relatively small amount of sample material was needed for the analysis, which makes this technique ideal for cell-specific analyses when the desired cells are limited in quantity.

We compared reproductive characteristics of the dendrobatid frog Colostethus trinitatis at the Tamana cave in the Central Range hills and at five forest stream sites in the Northern Range hills of Trinidad. There were significant differences between sites in adult female body size, intraovarian clutch size (in absolute terms and relative to body size) and the size of tadpoles carried by male frogs. Clutch sizes ranged from 6 to 13 in the Northern Range samples and from 12 to 26 at Tamana. Adult male body size was similar at all sites. Tadpoles carried by male C. trinitatis at Tamana were significantly smaller (mean length 14.2 mm) than those carried by males at the five Northern range sites (mean length 16.4–16.7 mm). The smallest clutches and largest tadpoles were found at Mount St Benedict, where there was evidence of size-selective predation on tadpoles by fish. Tamana, where clutches were large and tadpoles small, differed from the Northern Range sites in terms of the physical environment (cave vs forest) and the abundance of invertebrates. We discuss possible ultimate and proximate causes of the differences in clutch size and tadpole size, particularly size-selective predation and availability of food.

The phenology of calling activity and reproduction of a neotropical anuran community in French Guiana was studied during one rainy season. We investigated the correlation between calling activity, rainfall, temperature and water level in two ponds and recorded the occurrence of tadpoles of pond-breeding species. The study site contained 31 calling frog species, which were divided into groups according to reproductive mode. Increased rainfall was associated with increased reproductive activity in all groups, but temporal patterns in calling activity varied significantly between groups. Species with aquatic oviposition exhibited sporadic acoustic activity, aggregating into explosive breeding events following heavy rainfall. Species laying eggs in foam nests had the peak of calling activity at the start of the rainy season. Taxa with embryonic development on vegetation called mainly from middle to late wet season, being the only group which showed a significant correlation of calling with increasing water level. Dendrobatids with terrestrial oviposition and subsequent parental tadpole transportation were continuously active. Species with direct development or with non-feeding larvae were mainly active at the beginning of the rainy season. It is concluded that phenologies of calling activity in South American tropical anuran species are strongly influenced by abiotic factors like rainfall and availability of breeding sites. The temporal limitation of the rainy season forces species to adjust calling and reproductive activity according to their reproductive modes.

During a zoological survey in north-west Madagascar (Sahamalaza Peninsula) we documented aspects of the natural history of Boophis occidentalis (formerly a subspecies of B. albilabris and here raised to species level). Individual age assessed by skeletochronology ranged from 4 to 11 years. Breeding behaviour was observed at a seasonal stream after heavy rainfalls: five choruses of eight to 90 males aggregated in shallow brook sections. The males emitted low frequency calls and engaged in scramble battles. Only two females were found. A couple laid a large number of eggs, attached as a single layer on submerged stones. Tadpoles reared from these eggs had the typical morphology of brook-breeding Boophis species with a 1:5+5/3 keratodont formula and a relatively flattened body. Adult males had an anteroventrally oriented cloaca, a morphological trait shared with B. albilabris. Advertisement calls were similar in general structure to those of B. albilabris recorded at Anjanaharibe-Sud. They were unharmonious and consisted of pulsed notes emitted at irregular intervals. Much lower pulse rates in B. albilabris (30–38 pulses/s) than in B. occidentalis (90–109 pulses/s) support the specific distinctness of these taxa. The combination of characters related to reproductive biology in B. occidentalis and B. albilabris is unique in Boophis. The acontinuous breeding behaviour and large number of eggs per clutch are traits found in the pond-breeding species of the B. tephraeomystax group, while larval morphology and habitat are characteristic of brook-breeding species. Available morphological and molecular data place B. albilabris and B. occidentalis into the brook-breeding Boophis radiation. The acontinuous timing of reproduction and large egg number may be secondary adaptations that allowed the species to colonize arid regions of western Madagascar.

A new species of leptodactylid frog is described from Parque das Mangabeiras, in Belo Horizonte, Minas Gerais, south-eastern Brazil. The new species is a member of the Hylodes lateristrigatus group, and is characterized by medium size, snout rounded in dorsal view and protruding in lateral view, presence of vocal slits and sacs, upper surface of finger and toe discs with well-developed scutes, a weakly developed stripe on the upper lip, dorsum, arm, leg, and foot dark brown with small irregular brown and black dots, and absence of red and yellow spots in live specimens. The descriptions of the tadpoles and vocalizations, and information on natural history are provided. Hylodes perplicatus (Miranda-Ribeiro), previously considered a member of the H. nasus species group, is transferred to the H. lateristrigatus species group.