Immunological relationships of the South American frog genus Eupsophus (Leptodactylidae)

The agar-gel precipitin technique was used to examine the immunological relationships of the South American frog genus Eupsophus. This taxon shows similarities with the members of the Telmatobiini tribe and presents a characteristic precipitin band, here labelled as A.     

Size-dependent colouration balances conspicuous aposematism and camouflage

Colour is an important component of many different defensive strategies, but signal efficacy and detectability will also depend on the size of the coloured structures, and how pattern size interacts with the background. Consequently, size-dependent changes in colouration are common among many different species as juveniles and adults frequently use colour for different purposes in different environmental contexts. A widespread strategy in many species is switching from crypsis to conspicuous aposematic signalling as increasing body size can reduce the efficacy of camouflage, while other antipredator defences may strengthen. Curiously, despite being chemically defended, the gold-striped frog (Lithodytes lineatus, Leptodactylidae) appears to do the opposite, with bright yellow stripes found in smaller individuals, whereas larger frogs exhibit dull brown stripes. Here, we investigated whether size-dependent differences in colour support distinct defensive strategies. We first used visual modelling of potential predators to assess how colour contrast varied among frogs of different sizes. We found that contrast peaked in mid-sized individuals while the largest individuals had the least contrasting patterns. We then used two detection experiments with human participants to evaluate how colour and body size affected overall detectability. These experiments revealed that larger body sizes were easier to detect, but that the colours of smaller frogs were more detectable than those of larger frogs. Taken together our data support the hypothesis that the primary defensive strategy changes from conspicuous aposematism to camouflage with increasing size, implying size-dependent differences in the efficacy of defensive colouration. We discuss our data in relation to theories of size-dependent aposematism and evaluate the evidence for and against a possible size-dependent mimicry complex with sympatric poison frogs (Dendrobatidae).     

Antimicrobial,cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH₂), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp⁴→Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 μM) compared with the naturally occurring peptide. The substitution Ala¹⁸→Lys and the double substitution Asp⁴→Lys and Ala¹⁸→Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC₅₀ values in the range of 12–20 μM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC₅₀ = 20 μM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca²⁺] in BRIN-BD11 cells when incubated at a concentration of 1 μM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.     

Reproductive colonization of land by frogs: Embryos and larvae excrete urea to avoid ammonia toxicity

Vertebrate colonization of land has occurred multiple times, including over 50 origins of terrestrial eggs in frogs. Some environmental factors and phenotypic responses that facilitated these transitions are known, but responses to water constraints and risk of ammonia toxicity during early development are poorly understood. We tested if ammonia accumulation and dehydration risk induce a shift from ammonia to urea excretion during early stages of four anurans, from three origins of terrestrial development. We quantified ammonia and urea concentrations during early development on land, under well‐hydrated and dry conditions. Where we found urea excretion, we tested for a plastic increase under dry conditions and with ammonia accumulation in developmental environments. We assessed the potential adaptive role of urea excretion by comparing ammonia tolerance measured in 96h‐LC₅₀ tests with ammonia levels in developmental environments. Ammonia accumulated in foam nests and perivitelline fluid, increasing over development and reaching higher concentrations under dry conditions. All four species showed high ammonia tolerance, compared to fishes and aquatic‐breeding frogs. Both nest‐dwelling larvae of Leptodactylus fragilis and late embryos of Hyalinobatrachium fleischmanni excreted urea, showing a plastic increase under dry conditions. These two species can develop the longest on land and urea excretion appears adaptive, preventing their exposure to potentially lethal levels of ammonia. Neither late embryos of Agalychnis callidryas nor nest‐dwelling larvae of Engystomops pustulosus experienced toxic ammonia levels under dry conditions, and neither excreted urea. Our results suggest that an early onset of urea excretion, its increase under dry conditions, and elevated ammonia tolerance can all help prevent ammonia toxicity during terrestrial development. High ammonia represents a general risk for development which may be exacerbated as climate change increases dehydration risk for terrestrial‐breeding frogs. It may also be a cue that elicits adaptive physiological responses during early development.     

Ultrastructure of poison glands of South American frogs: a comparison between Physalaemus albonotatus and Leptodactylus chaquensis (Anura: Leptodactylidae)

Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead post-Golgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat "vacuolized" granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features.     

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.     

The frog fauna of Groote Eylandt, Northern Territory, Australia

The results are reported of a survey of the frog fauna of Groote Eylandt in the Gulf of Carpentaria in northern Australia. The fauna includes 13 species representing the families Hylidae and Leptodactylidae. The zoogeographic implications of the absence of certain species widely distributed on the mainland are discussed.