Revisiting current distribution and future habitat suitability models for the endemic Malabar Tree Toad (Pedostibes tuberculosus) using citizen science data.

Climate change is one of the major drivers of biodiversity loss. Among vertebrates, amphibians are one of the more sensitive groups to climate change due to their unique ecology, habitat requirements, narrow thermal tolerance and relatively limited dispersal abilities. We projected the influence of climate change on an endemic toad, Malabar Tree Toad (Pedostibes tuberculosus; hereafter MTT) from the Western Ghats biodiversity hotspot, India, for two different shared socio-economic pathways (SSP) using multiple modeling approaches for current and future (2061-2080) scenarios. The data used predominantly comes from a citizen science program, 'Mapping Malabar Tree Toad' which is a part of the Frog Watch citizen science program, India Biodiversity Portal. We also evaluated the availability of suitable habitats for the MTT in Protected Areas (PAs) under the current and future scenarios. Our results show that annual precipitation was the most important bioclimatic variable influencing the distribution of MTT. We used MaxEnt (MEM) and Ensemble (ESM) modeling algorithms. The predicted distribution of MTT with selected environmental layers using MEM was 4556.95 km2 while using ESM was 18,563.76 km2. Overlaying PA boundaries on predicted distribution showed 37 PAs with 32.7% (1491.37 km2) and 44 PAs with 21.9% (4066.25 km2) coverage for MEM and ESM respectively. Among eight future climate scenarios, scenarios with high emissions showed a decreased distribution range from 33.5 to 68.7% of predicted distribution in PAs, while scenarios with low emissions showed an increased distribution range from 1.9 to 111.3% in PAs. PAs from the Central Western Ghats lose most suitable areas with a shift of suitable habitats towards the Southern Western Ghats. This suggests that MTT distribution may be restricted in the future and existing PAs may not be sufficient to conserve their habitats. Restricted and discontinuous distribution along with climate change can limit the dispersal and persistence of MTT populations, thus enhanced surveys of MTT habitats within and outside the PAs of the Western Ghats are an important step in safeguarding the persistence of MTT populations. Overall, our results demonstrate the use of citizen science data and its potential in modeling and understanding the geographic distribution and the calling phenology of an elusive, arboreal, and endemic amphibian species.

Context-dependent effects of thermal acclimation on physiological correlates of animal personality in Asiatic toads.

Persistent individual variation in behaviour, or 'personality', is a widespread phenomenon in animals, and understanding the evolution of animal personality is a key task of current biology. Natural selection has been proposed to promote the integration of personality with animal 'intrinsic states', such as metabolic or endocrine traits, and this integration varies with ecological conditions. However, these external ecological modulatory effects have rarely been examined. Here, we investigate the effects of thermal acclimation on between-individual covariations between physiology and behaviour in Asiatic toads (Bufo gargarizans) along an altitudinal gradient. Our results reveal that the thermal modulatory effects on the covariations depend on the altitudinal population. Specifically, at low altitudes, between-individual covariations are highly plastic, with risk-taking behaviour covarying with baseline glucocorticoids (GCs) under warm acclimation, but risk-taking and exploration behaviour covarying with resting metabolic rate (RMR) under cold acclimation. In contrast, between-individual covariations are relatively fixed at high altitudes, with risk-taking behaviour consistently covarying with baseline GCs. Furthermore, at low altitudes, changes in covariations between RMR and personality are associated with adjustment of energy management models. Evidently, animal physiological states that determine or covary with personality can adapt according to the seasonal thermal environment and the thermal evolutionary background of populations. Our findings highlight the importance of a multi-system physiological approach to understand the evolution of animal personality.

Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

Transcription factor roles in the local adaptation to temperature in the Andean Spiny Toad Rhinella spinulosa.

Environmental temperature strongly influences the adaptation dynamics of amphibians, whose limited regulation capabilities render them susceptible to thermal oscillations. A central element of the adaptive strategies is the transcription factors (TFs), which act as master regulators that orchestrate stress responses, enabling species to navigate the fluctuations of their environment skillfully. Our study delves into the intricate relationship between TF expression and thermal adaptation mechanisms in the Rhinella spinulosa populations. We sought to elucidate the dynamic modulations of TF expression in prometamorphic and metamorphic tadpoles that inhabit two thermally contrasting environments (Catarpe and El Tatio Geyser, Chile) and which were exposed to two thermal treatments (25 °C vs. 20 °C). Our findings unravel an intriguing dichotomy in response strategies between these populations. First, results evidence the expression of 1374 transcription factors. Regarding the temperature shift, the Catarpe tadpoles show a multifaceted approach by up-regulating crucial TFs, including fosB, atf7, and the androgen receptor. These dynamic regulatory responses likely underpin the population's ability to navigate thermal fluctuations effectively. In stark contrast, the El Tatio tadpoles exhibit a more targeted response, primarily up-regulating foxc1. This differential expression suggests a distinct focus on specific TFs to mitigate the effects of temperature variations. Our study contributes to understanding the molecular mechanisms governing thermal adaptation responses and highlights the resilience and adaptability of amphibians in the face of ever-changing environmental conditions.

Morphology and vocalization comparison of the Houston Toad and the Dwarf American Toad: implications for their historic range.

Documenting changes in the distribution and abundance of a given taxon requires historical data. In the absence of long-term monitoring data collected throughout the range of a taxon, conservation biologists often rely on preserved museum specimens to determine the past or present, putative geographic distribution. Distributional data for the Houston Toad (Anaxyrus houstonensis) has consistently been confounded by similarities with a sympatric congener, the Dwarf American Toad (A. americanus charlesmithi), both in monitoring data derived from chorusing surveys, and in historical data via museum specimens. In this case, misidentification can have unintended impacts on conservation efforts, where the Houston Toad is federally endangered, and the Dwarf American Toad is of least concern. Previously published reports have compared these two taxon on the basis of their male advertisement call and morphological appearance, often with the goal of using these characters to substantiate their taxonomic status prior to the advent of DNA sequencing technology. However, numerous studies report findings that contradict one another, and no consensus on the true differences or similarities can be drawn. Here, we use contemporary recordings of wild populations of each taxon to test for quantifiable differences in male advertisement call. Additionally, we quantitatively examine a subset of vouchered museum specimens representing each taxon to test previously reported differentiating morphometric characters used to distinguish among other Bufonids of East-Central Texas, USA. Finally, we assemble and qualitatively evaluate a database of photographs representing catalogued museum vouchers for each taxon to determine if their previously documented historic ranges may be larger than are currently accepted. Our findings reveal quantifiable differences between two allopatric congeners with respect to their male advertisement call, whereas we found similarities among their detailed morphology. Additionally, we report on the existence of additional, historically overlooked, museum records for the Houston Toad in the context of its putative historic range, and discuss errors associated with the curation of these specimens whose identity and nomenclature have not been consistent through time. These results bookend decades of disagreement regarding the morphology, voice, and historic distribution of these taxa, and alert practitioners of conservation efforts for the Houston Toad to previously unreported locations of occurrence.

Dehydration followed by restraint sustains high circulating corticosterone and improves immunity in toads.

Amphibians are suffering population declines due to a variety of factors such as increased ultraviolet radiation, climate change, habitat loss, pathogens, and pollution, or a combination of these. Such changes are associated with a reduction in the availability of water, exposing these animals to a greater risk of desiccation. In this context, understanding how dehydration can modulate the hypothalamic-pituitary-interrenal axis (HPI) and the immune response is an imperative question to predict how stressors can affect amphibian species. We investigated whether dehydration promotes long-lasting effects on toads' ability to respond to a consecutive stressor (restraint) even if the toads are allowed to rehydrate, as well as its effects on the immune function. We also tested the hypothesis that the toads showing more severe dehydration would exhibit lower responsiveness to restraint challenge, even if the animals were allowed to rehydrate. Individuals of R. ornata were dehydrated mildly and severely. Thereafter, they were submitted to a restraint stress challenge for 1 and 24 h. Our results show that dehydration increased hematocrit and CORT in R. ornata toads. The restraint induced an acute stress response in fully hydrated toads (increased CORT and neutrophil: lymphocyte ratio). Otherwise, restraint in moistened cloth bags allowed full rehydration in previously dehydrated toads and did not induce an additional increase in CORT, but those toads sustained elevated CORT up to 24 h of restraint. Also, these animals showed increased neutrophil: lymphocyte ratio and the phagocytic activity of blood cells, even when they rehydrated during restraint. These results point to a continuous activation of the HPA during dehydration and subsequent restraint, even when they recovered from the dehydration state. Also, acute stressors seem to promote immune cell redistribution and augmentation of immune cellular function in R. ornata toads.

Runway extinction in terrestrial toads (Rhinella arenarum): Instrumental or Pavlovian?

Instrumental appetitive extinction involves the reduction of a previously reinforced response when its occurrence is no longer rewarded. Two experiments with terrestrial toads (Rhinella arenarum) tested whether the occurrence of a nonreinforced response is necessary for response extinction by varying the time of exposure to nonrewarded goal-box stimuli across groups. In Experiment 1, toads that received the same acquisition training (15 sessions, 1 session/day, 300 s of access to water in the goal box) were randomly assigned to two groups. In Group 600 (n=12), animals spent 600 s in the goal box in 8 daily extinction sessions (water present but inaccessible). In Group 0 (n=11), toads performed the runway response (i.e., walking from the start to the goal box) but were removed as soon as they entered the goal box, thus having minimal exposure to nonrewarded goal-box stimuli. The runway response was weakened in Group 600 across extinction trials, but exhibited little change in Group 0. In Experiment 2, toads were randomly assigned to two groups after the same acquisition training. Group 0 (n=7) was treated the same as Group 0 in the previous experiment. In Group RI (retention interval, n=7), toads remained in their home cage for 13 days. Finally, all animals received 4 extinction sessions with 300 s in the empty goal box. There was little behavioral change in Group 0 during the 13 sessions with minimal exposure to the goal box. In extinction, both groups reduced their runway response at similar rates. Although the procedures were instrumental, extinction of the runway response in toads can be accounted for in terms of a Pavlovian approach response to stimuli paired with reward and nonreward in the goal box.

Preparation for oxidative stress in Chinese toads (Bufo gargarizans) living under natural conditions along an altitudinal gradient.

Preparation for oxidative stress (POS) has been widely reported in animals under controlled laboratory conditions, but whether this phenomenon is visible in animals under natural conditions remains to be explored. Altitudinal gradients provide a good opportunity to address this question, since environmental conditions become more hostile with increasing altitude. Here, we investigated the levels of oxidative stress, oxidative damage, and antioxidant defenses in Chinese toads (Bufo gargarizans) along an altitudinal gradient (50 m, 1200 m, 2300 m, 3400 m above sea level). The results show that changing altitude led to a significantly lower ratio of oxidized to reduced glutathione in liver, with a higher value at 50 m. This ratio in muscle tissues did not differ significantly between altitudes of 50 m, 2300 m, and 3400 m. However, reduced glutathione content increased significantly along the altitude, with higher values in liver at 2300 m and higher values in skeletal muscle at 3400 m. Malondialdehyde (MDA) content in liver did not change significantly with increasing altitude. Brain and muscle tissues showed a higher MDA content at 50 m than the other three altitudes. The activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, as well as total antioxidant capacity, also displayed tissue-specific upregulation in heart, skeletal muscle, and brain, but all of these antioxidant enzymes except for glutathione-S-transferase were significantly reduced in liver along the altitudinal gradient. In summary, environmental factors at higher altitude did not lead to higher levels of oxidative stress and oxidative damage in B. gargarizans, mainly due to stronger antioxidant defenses. This study corroborates the occurrence of POS in high-altitude toads living under field conditions and contributes to revealing the biochemical adaptations to extreme environments at higher altitude.

The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species.

Baseline and stress-induced steroid plasma levels and immune function vary annually and are associated with vocal activity in male toads (Rhinella icterica).

Theoretical models predict that elevated androgen and glucocorticoid levels in males during the reproductive season promote immunosuppression. However, some studies report decreased stress response during this season. This study investigated annual variation in plasma corticosterone and testosterone levels, plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) in free-living male toads (Rhinella icterica). Toads were sampled in the field (baseline) and 1 h-post restraint over five months, and we considered the occurrence of vocal activity. Baseline corticosterone, testosterone, and BKA showed higher values during the reproductive period, specifically in calling male toads. The NLR was similar throughout the year, but higher values were observed in calling toads. Moreover, baseline NLR and BKA were positively correlated with both testosterone and corticosterone, suggesting higher steroid levels during reproduction are associated with enhanced cellular and humoral immunity. Despite fluctuation of baseline values, post-restraint corticosterone levels remained uniform over the year, indicating that toads reached similar maximum values throughout the year. Testosterone levels decreased following restraint before one specific reproductive period but increased in response to restraint during and after this period. Meanwhile, BKA decreased due to restraint only after the reproductive period, indicating immune protection and resilience to immunosuppression by stressors associated with steroid hormones during reproduction. Our results show that baseline and stress-induced hormonal and immune regulation varies throughout the year and are associated with vocal activity in R. icterica males, indicating a possible compromise between steroids and immune function in anuran males.

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.