Grouping rule in tadpole: is quantity more or size assortment more important?

The ability to perceive group size and discriminate the ontogeny of conspecifics would play a crucial role in the grouping behavior of animals. However, the relative importance of numerical quantity and size-assortative preferences in shaping grouping rules remains poorly understood. In this study, I examined the responses of Miyako toad (Bufo gargarizans miyakonis) tadpoles to number quantity and size discrimination by choice tests at different ontogenetic stages (small, medium, and large). The results revealed that small-sized tadpoles in early developmental stages significantly preferred larger numbers (4) compared to smaller ones (1). However, this preference was not observed in later developmental stages (medium and large). And interestingly, when there was no quantity bias, size discrimination was not observed in tadpoles, irrespective of their ontogeny. These findings suggest that Miyako toad tadpoles discern quantity, i.e., the number of conspecifics, but exhibit ontogeny-dependent utilization of this ability. Understanding the interplay between numerical quantity and size-assortative preferences in grouping behavior will provide esteemed insights into the adaptive value of number sense in vertebrates and shed light on evolutionary processes.

Exposure, but not timing of exposure, to a sulfonylurea herbicide alters larval development and behaviour in an amphibian species.

Environmental contamination is one of the major causes of biodiversity loss. Wetlands are particularly susceptible to contamination and species inhabiting these habitats are subjected to pollutants during sensitive phases of their development. In this study, tadpoles of a widespread amphibian, the spined toad (Bufo spinosus), were exposed to environmental concentrations of nicosulfuron (0 µg/L; 0.15 ± 0.05 µg/L and 0.83 ± 0.04 µg/L), a sulfonylurea herbicide, during different phases of development. Tadpoles were exposed during embryonic (12.98 ± 0.90 days) or larval development (93.74± 0.85 days), or throughout both phases, and we quantified development duration, morphological traits and behavioural features as responses to exposure. Developing tadpoles exposed to nicosulfuron were larger, but with smaller body, and had shorter but wider tail muscles. They were also more active and swam faster than control tadpoles and showed diverging patterns of behavioural complexity. We showed that higher concentrations had greater effects on individuals than lower concentrations, but the timing of nicosulfuron exposure did not influence the metrics studied: Exposure to nicosulfuron triggered similar effects irrespective of the developmental stages at which exposure occurred. These results further indicate that transient exposure (e.g., during embryonic development) can induce long-lasting effects throughout larval development to metamorphosis. Our study confirms that contaminants at environmental concentrations can have strong consequences on non-target organisms. Our results emphasize the need for regulation agencies and policy makers to consider sublethal concentrations of sulfonulyrea herbicides, such as nicosulfuron, as a minimum threshold in their recommendations.

Ontogeny of the middle ear and auditory sensitivity in the Natterjack toad (Epidalea calamita).

In most anuran amphibians, acoustic communication is of prime importance for mate localization and selection. The tympanic middle ear increases auditory sensitivity and directionality and is therefore expected to be favoured by natural selection. However, especially within the family of true toads (Bufonidae) there is a tendency for species to lose parts of the middle ear apparatus and consequently have a reduced sensitivity to high-frequency sounds (above 1 kHz). Part of the explanation for this may be that development of the middle ear is especially slow in bufonids, and thus the middle ear would be more likely to be lost or non-functional in paedomorphic species. However, a timeline of development of the middle ear has not been established previously. The goal of the present study was to investigate middle ear development in a toad species that has a well-known natural history and acoustic communication behaviour. We made a detailed study of anatomy and biophysics of the middle ear with measurements of auditory sensitivity across age in post-metamorphic natterjack toads (Epidalea calamita). The tadpoles and toadlets were raised in the laboratory, so their exact age was known, their auditory sensitivity was measured using auditory brainstem responses, and middle ear development and function were assessed by anatomical studies and laser vibrometry. We found that the developmental stage of the middle ear depends on the size of the toad rather than its age. The middle ear was functional at the earliest at a snout-vent length of 40 mm, which for these toads was around 500 days post-metamorphosis, close to the time of first reproduction. The functional, adult-like middle ear was shown to have 30 dB increased sensitivity to the dominant frequency of the mating call compared with sensitivities measured in newly metamorphosed individuals.

Fat rather than health - Ecotoxic responses of Bufo raddei to environmental heavy metal stress during the non-breeding season.

The non-breeding season is a critical period for iteroparous animals to repair damage and store energy, which is crucial for future survival and reproductive success. However, it is unknown how animals allocate energy efficiently among reproduction, self-maintenance, and repair of oxidative damage caused by breeding during the non-breeding period, particularly under pollution. In the present study, the self-maintenance response and reproductive strategy of Bufo raddei to long-term environmental heavy metal stress was explored during the non-breeding season. Heavy metal enrichment level, organ coefficients, nutritional status, gonadal developmental level, oxidation level, and the immune status of B. raddei during the non-breeding season were tested, energy allocation preferences and energy consumption costs were analyzed. The results revealed significant heavy metal accumulation through biomagnification in the organs of B. raddei from the polluted area. Under long-term environmental heavy metal pollution, the energy investment by B. raddei for growth and energy storage was higher than that for health-maintenance during the early life cycle stage. The energy inputted for immune and antioxidant functions was significantly lower, and the energy inputted for self-maintenance during the early life stage was significantly higher than that during the late stage. B. raddei from a heavy metal polluted area spent more energy consumption cost on immunity and oxidative stress, but suffered higher oxidative stress and lower immune status. Moreover, the reproductive input of individuals in a heavy metal polluted area was generally low during the non-breeding season, and the energy input for reproduction limited the energy input for self-maintenance in females.

Temperature regulation in the Balkan spadefoot (Pelobates balcanicus Karaman, 1928) at the beginning of nocturnal activity.

On land, the amphibians interact with the environment in a complex way-even small changes in the physiological conditions may significantly impact the behaviour and vice versa. In ectothermic tetrapods, the transition from inactive to active phase may be related to important changes in their thermal status. We studied the thermal ecology of adult Balkan spadefoots (Pelobates balcanicus Karaman, 1928) in northeastern Bulgaria. These toads spend the daytime buried between 10 and 15 cm in sandy substrates, and emerge after sunset. On the substrate, their thermal energy exchange is defined by the absence of heat flow from the sun. Secondary heat sources, like stored heat and infrared radiation from the soil play an important role for the thermal balance of the active spadefoot toads. At the beginning of their daily activity, we measured substrate temperature (at a depth of 11-12 cm), toad's surface body temperature, and also provided thermal profiles of the animals and the substrate surface in their microhabitats. In animals which recently emerged from the substrate, the temperature was comparatively higher and was closer to that of the subsoil on the spot. After that, body temperature decreased rapidly and continued to change slowly, in correlation with air temperature. We detected a temperature gradient on the dorsal surface of the toads. On the basis of our measurements and additional data, we discuss the eventual role of air humidity and the effects of surface and skin water evaporation on the water balance and activity of the investigated toads.

The Impacts of Transdermal Application of Corticosterone on Toad (Rhinella icterica) Immunity.

Recent studies have shown that acute physiological increases in endogenous glucocorticoid levels have immunostimulatory effects. Although post-acute stress immunosuppressive effects have also been described, the difference between enhancing and suppressing the immune response seems mediated by the stressor's duration, intensity, and the immune component under analysis. To elicit physiologically relevant corticosterone levels that can be found in Rhinella icterica toads after stressful events (e.g., restraint or captivity) and understand how acute increased glucocorticoid levels of different intensities affect corticosterone and testosterone plasma levels and immune parameters (in vitro plasma bacterial killing ability, neutrophil-to-lymphocyte ratio, and in vivo phagocytosis of peritoneal leukocytes), we submitted toads to the transdermal application of two corticosterone doses (1 and 10 µg). Corticosterone transdermal application increased corticosterone plasma levels with different intensities: 3 times for 1 µg and fourteen times for 10 µg, compared to the vehicle, and the neutrophil-to-lymphocyte ratio increased regardless of the corticosterone dose. However, there was no effect on testosterone levels and bacterial killing ability. Interestingly, both corticosterone doses promoted immunosuppression, decreasing peritoneal leukocytes' phagocytosis activity by 60% for toads receiving the dose of 1µg and 40% for those receiving 10 µg. Our results show the complexity of the relationship between increased corticosterone levels and immunomodulation. The different corticosterone doses promoted increases of distinct magnitudes in corticosterone plasma levels, with the less intense increase in corticosterone levels generating greater cell-mediated immunosuppression. Future studies using different corticosterone doses to achieve and compare physiological vs. pharmacological hormone levels are imperative to understanding these interrelationships between corticosterone and immune response.

Immune and endocrine responses of Cururu toads (Rhinella icterica) in their natural habitat after LPS stimulation.

Glucocorticoids and melatonin display immunomodulatory functions, with both immune-stimulatory and suppressor effects, depending on the context. While their immune properties are well-explored in mammals, there are still few studies on this immune-endocrine interaction in an inflammatory context in amphibians, all of them under captivity conditions, which can constitute a stressor for these animals. Evaluating how amphibians react to an immune challenge in the field would reveal relevant information regarding how immune-physiological parameters are modulated in natural conditions. This study aimed to investigate the effects of lipopolysaccharide (LPS) injection in male toads (Rhinella icterica) recently captured in their natural habitat in the Atlantic Forest at two different times of the day. We evaluated: splenic cytokines mRNA (interleukin [IL]-1ß, IL-6, IL-10, interferon-γ) and complement system protein (C1s), plasma bacterial killing ability (BKA), plasma corticosterone (CORT), melatonin (MEL), and testosterone (T) levels, and neutrophil to lymphocyte ratio (NLR), two hours post-injections. LPS-injection increased NLR, the gene expression of IL-1ß, and less evidently CORT levels independently of the time of the day. These results evidence LPS-induced inflammation, similarly observed in toads in captivity. Saline and LPS-injected toads showed a positive correlation between IL-1ß and IL-6, both cytokines with pro-inflammatory effects. Also, CORT was negatively associated with T, suggesting inhibition of the hypothalamus-pituitary-gonadal axis in the LPS-stimulated group. Our results are associated with the first stage of the inflammatory assemblage. Studies evaluating further steps of this process might lead to a better understanding of the immune-endocrine relations in amphibians.

The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm.

For amphibian species that display external fertilization in an aquatic environment, hypoosmotic shock to sperm cells can quickly result in damage to cellular structure and function. This study sought to determine how fertilization media osmolality, temperature, and time impact the stability of the mitochondrial vesicle's association with the sperm head and thus motility and quality of forward progression. The presence of the mitochondrial vesicle and its relationship with sperm motility and quality of forward progression were analyzed in sperm samples from the Fowler's toad (Anaxyrus fowleri) (n = 10) when held for six hours under two temperatures and four osmolalities. Results indicated that the presence of the mitochondrial vesicle is needed for sperm motility over time (p < 0.001), where higher osmolalities (p < 0.001) and lower temperatures (p < 0.001) correlated with maintaining the vesicle attachment to the spermatozoa. The higher osmolality of spermic urine was the most important factor for maintaining higher quality of forward progressive motility (p < 0.01) of spermatozoa. Sperm samples held at 4 °C and 40 mOsm/kg had the longest half-life for motility (540 min) and quality of forward progression (276 min), whereas sperm held at 22 °C and 2.5 mOsm/kg had the shortest half-life for motility (7 min) and quality of forward progression (18 min). Special attention should be given to the osmolality and temperature of fertilization solutions, or breeding tank water, when developing cold storage protocols for toad sperm or reproducing animals to ensure the retention of the mitochondrial vesicle for maximum fertilization capability.

Development of assisted reproductive technologies for the conservation of Atelopus sp. (spumarius complex).

Amphibians are in peril, given the ongoing sixth mass extinction of wildlife. Thus, Conservation Breeding Programs (CBPs) are attempting to breed some species under laboratory conditions. The incorporation of assisted reproduction technologies (ARTs), such as hormonal stimulation, sperm collection and cryopreservation, and in vitro fertilization is contributing to successful CBPs. The objective of this study was to apply ARTs in sexually mature individuals of an undescribed species of Atelopus (spumarius complex) (harlequin frog). Our procedure involves hormonal induction of gametogenesis in this species. We were able to induce gamete release through administration of human chorionic gonadotropin (hCG) in males, and in females this has been achieved through the sequential administration of hCG (priming doses), and combinations of hCG with gonadotropin releasing hormone analogue, GnRHa (ovulary dose). We standardized sperm cryopreservation by performing toxicity tests of cryoprotectants, fast/slow freezing and thawing, as well as supplementation of non-penetrating cryoprotectants (sugars). Next, we performed in vitro fertilization, evaluated the fertilization capacity of the cryopreserved sperm, and describe external features of fresh and cryopreserved sperm. We found that 10 IU/g hCG induced the release of the highest sperm concentrations between 3 and 5 h post-injection, while 2.5 IU/g hCG induced the release of eggs in most treated females. Under cryopreservation conditions, the highest recovery of forward progressive motility or FPM was 26.3 ± 3.5%, which was obtained in cryosuspensions prepared with the 5% DMF and 2.5% sucrose. Cryopreserved sperm showed narrower mitochondrial vesicles after thawing, while in frozen samples without cryodiluent showed 31% of spermatozoa lost their tails. In most cases, our attempts of in vitro fertilization were successful. However, only ∼10% of embryos were viable. Overall, our study demonstrates that the development of ARTs in individuals of Atelopus sp. (spumarius complex) bred in laboratory can be successful, which result in viable offspring through in vitro fertilization. Our study provides a baseline for assisted breeding protocols applicable to other harlequin frogs of the genus Atelopus.

Spermiation response to exogenous hormone therapy in hibernated and non-hibernated boreal toads (Anaxyrus boreas boreas).

Conservation programs for threatened high- elevation amphibian species rely on hibernation to trigger appropriate male reproductive behaviours and gametogenesis. Although common practice and anecdotal observations have supported the practice of hibernation, there is limited empirical evidence documenting the effects on reproduction in these species. In this study, the effect of hibernation on sperm quantity and quality was evaluated for the alpine species Anaxyrus boreas boreas . Hibernated (n =19) and non-hibernated (n =21) male toads were administered 10IUg-1 body weight (BW) human chorionic gonadotropin (hCG) and spermic urine was collected over 24h. Hibernation had no effect on the number of males undergoing spermatogenesis, but hibernated males produced sperm in higher concentrations. Sperm quality was measured in terms of total motility, forward progressive motility and quality of forward progression. Although there was no difference in the total sperm motility of samples from hibernated and non-hibernated toads, the percentage of sperm exhibiting forward progressive motility and the quality of forward progression was significantly greater from hibernated toads. These results support our hypothesis that hibernation impacts both sperm quantity and quality in male boreal toads. This study will better inform captive breeding management decisions for threatened alpine species, in imminent danger of extinction.

Increased rates of dispersal of free-ranging cane toads (Rhinella marina) during their global invasion.

Invasions often accelerate through time, as dispersal-enhancing traits accumulate at the expanding range edge. How does the dispersal behaviour of individual organisms shift to increase rates of population spread? We collate data from 44 radio-tracking studies (in total, of 650 animals) of cane toads (Rhinella marina) to quantify distances moved per day, and the frequency of displacement in their native range (French Guiana) and two invaded areas (Hawai'i and Australia). We show that toads in their native-range, Hawai'i and eastern Australia are relatively sedentary, while toads dispersing across tropical Australia increased their daily distances travelled from 20 to 200 m per day. That increase reflects an increasing propensity to change diurnal retreat sites every day, as well as to move further during each nocturnal displacement. Daily changes in retreat site evolved earlier than did changes in distances moved per night, indicating a breakdown in philopatry before other movement behaviours were optimised to maximise dispersal.

Toads use the subsurface thermal gradient for temperature regulation underground.

As ectotherms with moist, permeable skins, amphibians continually seek a physiological balance between maintaining hydration and optimizing body temperature. Laboratory studies have suggested that dehydrated and starved amphibians should select cooler temperatures to slow the rate of water loss and reduce metabolism. However, much less is known about amphibian thermoregulatory behaviour in the wild, where environmental conditions and constraints may be more variable. In seasonally cold environments, where animals must maximize growth, gamete production and/or fat storage for winter dormancy over a short active season, maintaining a high metabolic rate may be primary. We investigated the thermoregulatory behaviour of the Fowler's Toads, Anaxyrus fowleri, in the wild at their northern range limit at Long Point, Ontario. We outfitted adult toads with small temperature loggers and radio-tracked them for periods of 24 hours. Simultaneously, we also recorded air and subsurface temperatures to a depth of 18.6 cm. When active at night, toads rapidly equilibrated with ambient air temperatures. However, during the day, resting toads selected and maintained body temperatures around 30 °C during the heat of the day by adjusting the depth to which they were buried. This strongly suggests that they behaviourally thermoregulate during their resting hours to maintain a high metabolic rate without regard to the dryness of their immediate surroundings.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality.

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species' reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Infection Status as the Basis for Habitat Choices in a Wild Amphibian.

AbstractAnimals challenged with disease may select specific habitat conditions that help prevent or reduce infection. Whereas preinfection avoidance of habitats with a high risk of disease exposure has been documented in both captive and free-ranging animals, evidence of switching habitats after infection to support the clearing of the infection is limited to laboratory experiments. The extent to which wild animals proximately modify habitat choices in response to infection status thus remains unclear. We investigated preinfection behavioral avoidance and postinfection habitat switching using wild, radio-tracked boreal toads (Anaxyrus boreas boreas) in a population challenged with Batrachochytrium dendrobatidis (Bd), a pathogenic fungus responsible for a catastrophic panzootic affecting hundreds of amphibian species worldwide. Boreal toads did not preemptively avoid microhabitats with conditions conducive to Bd growth. Infected individuals, however, selected warmer, more open habitats, which were associated with elevated body temperature and the subsequent clearing of infection. Our results suggest that disease can comprise an important selective pressure on animal habitat and space use. Habitat selection models, therefore, may be greatly improved by including variables that quantify infection risk and/or the infection status of individuals through time.

Cadmium induced skeletal underdevelopment, liver cell apoptosis and hepatic energy metabolism disorder in Bufo gargarizans larvae by disrupting thyroid hormone signaling.

Cadmium (Cd) is hazardous to human health and it is also highly detrimental to amphibian life. In this study, Bufo gargarizans larvae were exposed to environmentally relevant Cd concentrations of 5, 100 and 200 µg L-1 from Gosner stage (Gs) 26 to Gs 42 of metamorphic climax about 6 weeks. The results showed thyroid structural injuries and thyroid signaling disruption were induced by high Cd exposure (100 and 200 µg L-1). Moreover, tadpole skeleton including whole body, vertebrata, forelimb and hindlimb was developmentally delayed by high Cd exposure through downregulating the mRNA expressions of genes involved with skeletal ossification and growth pathway. Moreover, liver histopathological injuries were caused by high Cd exposure featured by hepatocytes malformation, nuclear degeneration and increasing melanomacrophage centers. Meanwhile, liver apoptosis rate showed on the rise in a dose-dependent way and Cd stimulated liver apoptosis by upregulating mRNA expressions of genes related to extrinsic and intrinsic apoptosis pathways. Furthermore, high Cd caused hepatic glucometabolism disorder by decreasing the genetic expressions associated with glycolysis and mitochondrial oxidative phosphorylation. In addition, liver lipid metabolism was disrupted by high Cd exposure through downregulating mRNA levels of genes related to fatty oxidation and upregulating mRNA levels of genes related to fatty acid synthesis. We suggested that Cd did great harm to tadpole health by disturbing thyroid function, skeletal growth, liver cell apoptosis signaling and hepatic energy metabolism pathway.

Brain profiling of endogenous Neuropeptide Y (NPY) in distinct reproductive phases of adult male Microhyla ornata.

Neuropeptide Y(NPY) is known to play a pivotal role in various physiological functions including appetite and reproduction. While studies in mammals, fishes and reptiles suggest a temporal and evolutionary conserved role of NPY, the information in amphibian is scanty. We have investigated the reproductive phase related variations of NPY in the brain of Microhyla ornata (M. ornata), using immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR). The highest expression of NPY peptide was observed in the preoptic area (Poa), nucleus infundibularis ventralis (NIV) and nucleus reticularis isthmi (NRIS) of M. ornata in breeding season compared to pre-breeding as well as post-breeding season. In parallel, highest mRNA levels of NPY were also observed in the breeding season in the middle region of brain that includes hypothalamus of M. ornata. Variation in the levels of NPY peptide and mRNA levels in the brain of M. ornata point towards seasonal control of appetite and reproduction.

Challenges of a novel range: Water balance, stress, and immunity in an invasive toad.

Species introduced by human activities can alter the normal functioning of ecosystems promoting negative impacts on native biodiversity, as they can rapidly expand their population size, demonstrating phenotypic plasticity and possible adaptive capacity to novel environments. Twenty years ago, the guttural toad, Sclerophrys gutturalis, was introduced to a peri-urban area of Cape Town, with cooler and drier climatic characteristics than its native source population, Durban, South Africa. Our goal was to understand the phenotypic changes, in terms of physiology and immunity, of populations in native and novel environments. We evaluated body index (BI), field hydration level, plasma corticosterone levels (CORT), proportion of neutrophils: lymphocytes (N: L), plasma bacterial killing ability (BKA), and hematocrit (HTC) in the field, and after standardized stressors (dehydration and movement restriction) in males from the native and invasive populations. Toads from the invasive population presented lower BI and tended to show a lower field hydration state, which is consistent with living in the drier environmental conditions of Cape Town. Additionally, invasive toads also showed higher BKA and N:L ratio under field conditions. After exposure to stressors, invasive animals presented higher BKA than the natives. Individuals from both populations showed increased CORT after dehydration, an intense stressor for these animals. The highest BKA and N:L ratio in the field and after submission to stressors in the laboratory shows that the invasive population has a phenotype that might increase their fitness, leading to adaptive responses in the novel environment and, thus, favoring successful dispersion and population increase.

Combining spawn egg counts, individual photo-ID and genetic fingerprinting to estimate the population size and sex ratio of an endangered amphibian.

According to the International Union for Conservation of Nature Red List, 41% of the world's amphibian species are threatened with extinction, making them more threatened than any other vertebrate group nowadays. Given the global amphibian crisis, comprehensive understanding of demographics and population trends of declining and threatened species is essential for effective management and conservation strategies. Counting egg spawns is widely used to assess population abundance in pond breeding anurans. However, it is unknown how such counts translate into robust population size estimations. We monitored the breeding activity of the Natterjack toad (Epidalea calamita), combining egg string counts and individual photo-identification with Capture-Mark-Recapture population size and operational sex ratio estimation. Male Natterjack toads were identified by the pattern of natural markings with repeated ID of the same individual confirmed for 10% of the samples using genetic fingerprinting. We identified 647 unique individuals within a closed study population at Caherdaniel, Co Kerry. Population estimates derived from egg string counts estimated a breeding population of 368 females (95% CI 353-384) and Capture-Mark-Recapture estimated a breeding population of 1698 males (95% CI 1000-2397). The female:male sex ratio was conservatively estimated at 1:5 (95% CI 1:3-1:6) where 62% ± 6% of females were assumed to spawn. These substantially departed from any priori assumption of 1:1 which could have underestimated the breeding population by up to 72%. Where amphibian absolute population size estimation is necessary, methods should include empirical survey data on operational sex ratios and not rely on assumptions or those derived from the literature which may be highly population and/or context-dependent.

Variation in Bufadienolide Composition of Parotoid Gland Secretion From Three Taxa of Japanese Toads.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Feeding alters the preferred body temperature of Cururu toads, Rhinella diptycha (Anura, Bufonidae).

Ectothermic organisms depend primarily on external heat sources and behavioural adjustments to regulate body temperature. Under controlled conditions, in a thermal gradient, body temperature often clusters around a more or less defined range of preferred body temperatures (Tpref). However, Tpref may be modified in response to environmental parameters and/or physiological state. For example, meal ingestion is sometimes followed by a post-prandial thermophilic response leading to a transient increment in Tpref. Although thought to optimize digestive processes, its occurrence, magnitude, and possible determinants remains scarcely documented for anuran amphibians. Herein, we investigated whether the Cururu toad, Rhinella diptycha, exhibits a post-prandial thermophilic response by monitoring the body temperature of fasting and fed toads while they were maintained in a thermal gradient. We found that the toads' Tpref increased by about 13% from day 2 to 4 after feeding, in comparison with the Tpref recorded under fasting. Also, fed animals exhibited a broader range for Tpref at days 2 and 3 post-prandial, which reflects a greater level of locomotor activity compared to fasting individuals. We conclude that R. diptycha is capable to exhibit a post-prandial thermophilic response under the controlled conditions of a thermal gradient. Although this thermoregulatory adjustment is thought to optimize meal digestion yielding important energetic and ecological benefits, its occurrence in anuran amphibians in nature remains uncertain.