Bifenthrin induces changes in clinical poisoning symptoms, oxidative stress, DNA damage, histological characteristics, and transcriptome in Chinese giant salamander (Andrias davidianus) larvae.

Bifenthrin (BF) is a broad-spectrum insecticide that has gained widespread use due to its high effectiveness. However, there is limited research on the potential toxic effects of bifenthrin pollution on amphibians. This study aimed to investigate the 50 % lethal concentration (LC50) and safety concentration of Chinese giant salamanders (CGS) exposed to BF (at 0, 6.25,12.5,25 and 50 µg/L BF) for 96 h. Subsequently, CGS were exposed to BF (at 0, 0.04, and 4 µg/L BF) for one week to investigate its toxic effects. Clinical poisoning symptoms, liver pathology, oxidative stress factors, DNA damage, and transcriptome differences were observed and analyzed. The results indicate that exposure to BF at 4 µg/L significantly decreased the adenosine-triphosphate (ATP), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) contents in the brain, liver, and kidney of CGS. Additionally, the study found that the malondialdehyde (MDA), reactive oxygen species (ROS), and 8-hydroxydeoxyguanosine (8-OHdG) contents were increased. The liver tissue exhibited significant inflammatory reactions and structural malformations. RNA-seq analysis of the liver showed that BF caused abnormal antioxidant indices of CGS. This affected molecular function genes such as catalytic activity, ATP-dependent activity, metabolic processes, signaling and immune system processes, behavior, and detoxification, which were significantly upregulated, resulting in the differential genes significantly enriched in the calcium signaling pathway, PPARα signaling pathway and NF-kB signaling pathway. The results suggest that BF induces the abnormal production of free radicals, which overwhelms the body's self-defense system, leading to varying degrees of oxidative stress. This can result in oxidative damage, DNA damage, abnormal lipid metabolism, autoimmune diseases, clinical poisoning symptoms, and tissue inflammation. This work provides a theoretical basis for the rational application of bifenthrin and environmental risk assessment, as well as scientific guidance for the conservation of amphibian populations.

Linear and Nonlinear Behaviors of the Photoreceptor Coupled Network.

Photoreceptors are electrically coupled to one another, and the spatiotemporal properties of electrical synapses in a two-dimensional retinal network are still not well studied, because of the limitation of the single electrode or pair recording techniques which do not allow simultaneously measuring responses of multiple photoreceptors at various locations in the retina. A multiple electrode recording system is needed. In this study, we investigate the network properties of the two-dimensional rod coupled array of the salamander retina (both sexes were used) by using the newly available multiple patch electrode system that allows simultaneous recordings from up to eight cells and to determine the electrical connectivity among multiple rods. We found direct evidence that voltage signal spread in the rod-rod coupling network in the absence of I h (mediated by HCN channels) is passive and follows the linear cable equation. Under physiological conditions, I h shapes the network signal by progressively shortening the response time-to-peak of distant rods, compensating the time loss of signal traveling from distant rods to bipolar cell somas and facilitating synchronization of rod output signals. Under voltage-clamp conditions, current flow within the coupled rods follows Ohm's law, supporting the idea that nonlinear behaviors of the rod network are dependent on membrane voltage. Rod-rod coupling is largely symmetrical in the 2D array, and voltage-clamp blocking the next neighboring rod largely suppresses rod signal spread into the second neighboring rod, suggesting that indirect coupling pathways play a minor role in rod-rod coupling.

Evaluating translocation success of wild eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Blue Ridge Ecoregion streams using pre- and post-translocation home range sizes and movement metrics.

Translocations of freshwater species have become a widespread conservation strategy to mitigate the impacts of habitat fragmentation, yet they are not often rigorously monitored using animal movement data to determine their success. We demonstrate the value of monitoring pre- and post-translocation movements and home-range sizes of a fully-aquatic, benthic stream salamander, the eastern hellbender (Cryptobranchus a. alleganiensis) to determine translocation success. We studied the home range sizes, movements, and habitat use of individuals (n = 27) in two self-sustaining populations (S1 & S2) for one year, and then subsequently collected similar data from a subset of these individuals (n = 17) that were translocated into two nearby streams (T1 & T2) with dam-isolated, declining populations in the Blue Ridge Ecoregion of Tennessee. We collected 1,571 location data points (869 pre-translocation and 715 post-translocation) from four study sites, and evaluated effects of mass, sex, and pre-translocation home range size/sedentariness, as well as habitat covariates on home range size and movements. Hellbender home range sizes increased from pre-translocation estimates at both sites, but response depended primarily on physical characteristics of release sites. Home range and fine-scale movement metrics indicated that hellbenders translocated from S1 to T1 settled in more quickly, had greater site fidelity, and smaller home ranges than hellbenders translocated from S2 to T2. Hellbender movements were influenced by cover rock size and density rather than individual characteristics. Study-long survival rates of translocated hellbenders increased from S1 to T1 (80% to 100%) and decreased from S2 to T2 (76% to 33%). Monitoring pre- and post-translocation movements was a valuable method for evaluating short-term translocation success in a freshwater environment. For future hellbender translocations, managers should prioritize selecting suitable release sites with contiguous boulder-dense areas (1-2 per m2), adequate prey (crayfish) densities (>1/m2), and habitats with low risk of predation.

Disease state associated with chronic toe lesions in hellbenders may alter anti-chytrid skin defenses.

Hellbenders (Cryptobranchus alleganiensis) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans (Bsal) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal. Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd. MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects of disease associated with toe lesions.

Developmental switch from morphological replication to compensatory growth for salamander lung regeneration.

Salamanders possess a pair of lungs for active air breathing, but the lung respiration is fully operational only during the late stage of development, particularly after metamorphosis. Larval salamanders mainly exchange air through the gills and skin, thus sparing the developing lungs. Salamanders can repair their lungs after injury, but a comparative analysis of regenerative responses between the lungs of young and adult animals is lacking. In this study, lung resections were performed in both larval and adult newts (Pleurodeles waltl). The cellular dynamics, tissue morphology and organ function during lung regeneration were examined and the Yap mutants were produced with CRISPR tools. We found that salamander switches the regenerative strategies from morphological replication through the blastema formation to compensatory growth via resident epithelial cells proliferation upon pulmonary resection injury as it transitions beyond metamorphosis. The larval animals achieve lung regeneration by forming a transient blastema-like structure and regrowing full-sized developing lungs, albeit unventilated. The adults repair injured lungs via massive proliferating epithelial cells and by expanding the existing alveolar epithelium without neo-alveolarization. Yap signalling promotes epithelial cell proliferation and prevents epithelial-to-mesenchymal transition to restore functional respiration. The salamanders have evolved distinct regenerative strategies for lung repair during different phases of life. Our results demonstrate a novel strategy for functional lung recovery by inducing epithelial cell proliferation to strengthen the remaining alveoli without rebuilding new alveoli.

Immunohematological Parameters of Ectothermal Amphibians of the Fauna of the Middle Urals: Siberian Salamander Salamandrella Keyserlingii Dybovsky, 1870 (Caudata) and Lake Frog Pelophylax Ridibundus Pallas, 1771 (Anura).

A comparative analysis of hematological parameters was for the first time performed in two ectothermal amphibians of the Middle Ural fauna, the Siberian salamander Salamandrella keyserlingii and lake frog Pelophylax ridibundиs. Species specificity of immune defense was demonstrated with respect to granulocyte and agranulocyte counts (p < 0.001). A high lymphocyte content (73.3-76.1%) of provides for the activation of acquired adaptive immunity mechanisms in the thermophilic lake frog. The Siberian salamander is adapted to low negative temperatures and has a set of nonspecific leukocytes (39.3-44.4%). Innate immunity is better developed in the Siberian salamander compared with the lake frog.

Gliding and parachuting by arboreal salamanders.

Wandering salamanders (Aneides vagrans) reside in the crowns of the world's tallest trees and have been observed to readily jump from the canopy when disturbed1,2. Here, we describe the aerial performance of falling A. vagrans, which maintain stable gliding postures via adjustments of the limbs and tail in lieu of specialized control surfaces. In wind tunnel trials, A. vagrans parachuted consistently and slowed their vertical speed by up to 10% while falling. Furthermore, A. vagrans coupled parachuting with parasagittal undulations of the tail and torso to effect gliding at non-vertical angles (minimum of ∼84°) in 58% of trials. Selection pressures imposed on falling from heights can be substantial, and have resulted in the evolution of diverse aerial behaviors among arboreal taxa; nonetheless, aerial behavior occurring in arboreal salamanders is surprising, and calls for further work on the natural occurrence of falling, gliding, and directed aerial descent in canopy-dwelling tetrapods.

Temperature and nutrient conditions modify the effects of phenological shifts in predator-prey communities.

Although there is mounting evidence indicating that the relative timing of predator and prey phenologies determines the outcome of trophic interactions, we still lack a comprehensive understanding of how the environmental context (e.g., abiotic conditions) influences this relationship. Environmental conditions not only frequently drive shifts in phenologies, but they can also affect the very same processes that mediate the effects of phenological shifts on species interactions. Therefore, identifying how environmental conditions shape the effects of phenological shifts is key to predicting community dynamics across a heterogeneous landscape and how they will change with ongoing climate change in the future. Here I tested how environmental conditions shape the effects of phenological shifts by experimentally manipulating temperature, nutrient availability, and relative phenologies in two predator-prey freshwater systems (mole salamander-bronze frog vs. dragonfly larvae-leopard frog). This allowed me to (1) isolate the effects of phenological shifts and different environmental conditions; (2) determine how they interact; and (3) evaluate how consistent these patterns are across different species and environments. I found that delaying prey arrival dramatically increased predation rates, but these effects were contingent on environmental conditions and the predator system. Although nutrient addition and warming both significantly enhanced the effect of arrival time, their effect was qualitatively different across systems: Nutrient addition enhanced the positive effect of early arrival in the dragonfly-leopard frog system, whereas warming enhanced the negative effect of arriving late in the salamander-bronze frog system. Predator responses varied qualitatively across predator-prey systems. Only in the system with a strong gape limitation were predators (salamanders) significantly affected by prey arrival time and this effect varied with environmental context. Correlations between predator and prey demographic rates suggest that this was driven by shifts in initial predator-prey size ratios and a positive feedback between size-specific predation rates and predator growth rates. These results highlight the importance of accounting for temporal and spatial correlations of local environmental conditions and gape limitation when predicting the effects of phenological shifts and climate change on predator-prey systems.

Are they arboreal? Climbing abilities and mechanics in the red-backed salamander (Plethodon cinereus).

While red-backed salamanders (Plethodon cinereus) are most often observed in terrestrial forested areas, several studies report arboreal substrate use and climbing behavior. However, salamanders do not have any of the anatomical features commonly observed in specialized climbing species (e.g., claws, setae, suction cups). Instead, salamanders cling to surfaces using the shear and adhesive properties of their mucous layer. In this study, we explore the capabilities and spatiotemporal gait patterns of arboreal locomotion in the red-backed salamander as they move across twelve substrate conditions ranging in diameter, orientation, and roughness. On arboreal substrates, red-backed salamanders decreased locomotor speed, stride frequency, phase and stride length, and increased duty factor and stride duration. Such responses have been observed in other non-salamander species and are posited to increase arboreal stability. Furthermore, these findings indicate that amphibian locomotion, or at least the locomotor behavior of the red-backed salamander, is not stereotyped and that some locomotor plasticity is possible in response to the demands of the external environment. However, red-backed salamanders were unable to locomote on any small-diameter or vertically-oriented coarse substrates. This finding provides strong evidence that the climbing abilities of red-backed salamanders are attributable solely to mucous adhesion and that this species is unable to produce the necessary external "gripping" forces to achieve fine-branch arboreal locomotion or scale substrates where adhesion is not possible. The red-backed salamander provides an ideal model for arboreal locomotor performance of anatomically-unspecialized amphibians and offers insight into transitionary stages in the evolution of arborealism in this lineage.

Rapid phenotypic change in a polymorphic salamander over 43 years.

Color polymorphic animals offer a unique system for studying intraspecific phenotypic responses to climate change. Discrete color morphs are easy to identify, and correlated trait responses of morphs can indicate how climate warming may facilitate long-term maintenance of polymorphisms. We use a historical dataset spanning 43 years to examine temporal shifts in color morph frequency and body size in response to climate in the Eastern Red-backed Salamander, Plethodon cinereus, which contains a widespread striped/unstriped color polymorphism. We created a pipeline to extract high-throughput trait data from fluid-preserved museum specimens where we batch-photographed salamanders, de-aggregated individual specimens from photographs, and solicited help of community scientists to score color morphs. We used a linear modeling framework that includes information about spatial population structure to demonstrate that color morph frequency and body size vary in response to climate, elevation, and over time, with an overall trend of higher frequency and decreased body size of the striped morph, but increased size of the unstriped morph. These surprising results suggest that morphs may be responding to multiple climate and geographic drivers through co-adapted morphological changes. This work highlights new practices of extracting trait data from museum specimens to demonstrate species phenotypes response to climate change.

Altered metabolic state impedes limb regeneration in salamanders.

Salamanders are unique among tetrapods in their ability to regenerate their limbs throughout life. Like other poikilothermic amphibians, salamanders also show a remarkable capacity to survive long periods of starvation. Whether the physiological reserves necessary for tissue regeneration are preserved or sacrificed in starved salamanders is unknown. In the current study, we maintained Iberian ribbed newts ( Pleurodeles waltl) under extreme physiological stress to assess the extent of regeneration and identify the molecular and cellular changes that may occur under such conditions. After 19 months of complete food deprivation, the animals exhibited extensive morphological and physiological adaptations but remained behaviorally active and vigilant. Autophagy was elevated in different tissues and the transformed gut microbiota indicated remodeling of the intestinal tract related to autophagy. Upon limb amputation in animals starved for 21 months, regeneration proceeded with progenitor cell proliferation and migration, leading to limb blastema formation. However, limb outgrowth and patterning were substantially attenuated. Blockage of autophagy inhibited cell proliferation and blastema formation in starved animals, but not in fed animals. Hence, tissue autophagy and the regenerative response were tightly coupled only when animals were under stress. Our results demonstrate that under adverse conditions, salamanders can exploit alternative strategies to secure blastema formation for limb regeneration.

Phenotypic Plasticity of Salamander Hatchlings in the Pre-Feeding Stage in Response to Future Prey.

Vulnerability of animals immediately after hatching may induce plasticity in early ontology that becomes important for subsequent survival and growth. Ezo salamanders (Hynobius retardatus) are amphibians inhabiting ponds in Hokkaido, Japan where ezo brown frogs (Rana pirica) spawn on occasion. The salamander larvae must achieve sufficient size in order to successfully capture frog tadpoles, and we examined whether the presence of tadpoles causes development of greater body and/or gape size in newly hatched salamander larvae, which will in turn result in advantageous future prey-predator interactions. To examine this hypothesis, we conducted three laboratory experiments to demonstrate the phenotypic plasticity of salamander hatchlings in response to the presence or absence of frog tadpoles and to screen the type of signals involved in the expression of the phenotypic plasticity. First, salamander hatchlings were reared alone or with tadpoles, and the growth and morphological traits of the hatchlings were compared. The results showed that hatchling larvae grew faster with a more developed gape in the presence of tadpoles. Next, to identify the type of signals inducing this plasticity, two separate experiments with manipulated chemical and visual signals from tadpoles were conducted. The findings showed that faster growth and a more developed gape were induced by chemical but not visual signals. This plasticity may be an adaptive strategy because it increases the likelihood of preying on tadpoles in future prey-predator interactions.

Pre-breeding androgen and glucocorticoid profiles in the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis).

Seasonally breeding species exhibit cyclical changes in circulating steroid hormone profiles that correspond with changes to their reproductive behavior and ecology. Such information is critical to the conservation of imperiled and data-deficient species, such as the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis). We determined changes in plasma testosterone (T), dihydrotestosterone (DHT), 11-ketotestosterone (11-KT), 11-ketoandrostenedione (11-KA), dehydroepiandrosterone (DHEA), cortisol, corticosterone, and progesterone (P4) during a four-month period preceding breeding in adult male and female eastern hellbenders. This pre-breeding period is characterized by increased diel movement and aggression by both sexes, follicular development and yolk production in females, and sperm production, territoriality, and nest site establishment in males. In both males and females, we observed a progressive increase in circulating T and DHT during the pre-reproductive season, both peaking in August (17 days before breeding), but concentrations of both hormones were higher in males. Conversely, 11-KT was higher in females, but did not vary significantly by date. These results suggest that T and DHT are the predominant androgens in eastern hellbenders and are likely important regulators of reproductive processes in both males and females. The detection of significant quantities of DHT and 11-KT in females is particularly interesting, considering that unlike T, neither of these androgens can be converted to estrogens. Therefore, it seems possible that aggression or some aspect of reproduction in the female eastern hellbender may be directly mediated by androgen signaling. Baseline cortisol did not vary throughout the pre-breeding period but was higher in females than males, and also became highly variable in females leading up to breeding. Progesterone, 11-KA, DHEA, and corticosterone were rarely or never detected, and thus, do not appear to be important during the pre-reproductive season. This study provides a physiological framework for future studies of hellbender reproductive biology, which could ultimately be important for their conservation.

Landscape resistance constrains hybridization across contact zones in a reproductively and morphologically polymorphic salamander.

Explicitly accounting for phenotypic differentiation together with environmental heterogeneity is crucial to understand the evolutionary dynamics in hybrid zones. Species showing intra-specific variation in phenotypic traits that meet across environmentally heterogeneous regions constitute excellent natural settings to study the role of phenotypic differentiation and environmental factors in shaping the spatial extent and patterns of admixture in hybrid zones. We studied three environmentally distinct contact zones where morphologically and reproductively divergent subspecies of Salamandra salamandra co-occur: the pueriparous S. s. bernardezi that is mostly parapatric to its three larviparous subspecies neighbours. We used a landscape genetics framework to: (i) characterise the spatial location and extent of each contact zone; (ii) assess patterns of introgression and hybridization between subspecies pairs; and (iii) examine the role of environmental heterogeneity in the evolutionary dynamics of hybrid zones. We found high levels of introgression between parity modes, and between distinct phenotypes, thus demonstrating the evolution to pueriparity alone or morphological differentiation do not lead to reproductive isolation between these highly divergent S. salamandra morphotypes. However, we detected substantial variation in patterns of hybridization across contact zones, being lower in the contact zone located on a topographically complex area. We highlight the importance of accounting for spatial environmental heterogeneity when studying evolutionary dynamics of hybrid zones.

Exposure to glucocorticoids alters life history strategies in a facultatively paedomorphic salamander.

Polyphenisms, where two or more alternative, environmentally-cued phenotypes are produced from the same genotype, arise through variability in the developmental rate and timing of phenotypic traits. Many of these developmental processes are controlled or influenced by endogenous hormones, such as glucocorticoids, which are known to regulate a wide array of vertebrate ontogenetic transitions. Using the mole salamander, Ambystoma talpoideum, as a model, we investigated the role of glucocorticoids in regulating facultative paedomorphosis, an ontogenetic polyphenism where individuals may delay metamorphosis into terrestrial adults. Instead, individuals reproduce as aquatic paedomorphic adults. Paedomorphosis often occurs when aquatic conditions remain favorable, while metamorphosis typically occurs in response to deteriorating or "stressful" aquatic conditions. Since glucocorticoids are central to the vertebrate stress response and are known to play a central role in regulating obligate metamorphosis in amphibians, we hypothesized that they are key regulators of paedomorphic life history strategies. To test this hypothesis, we compared development of larvae in outdoor mesocosms exposed to Low, Medium, and High exogenous doses of corticosterone (CORT). Results revealed that body size and the proportion of paedomorphs were both inversely proportional to exogenous CORT doses and whole-body CORT content. Consistent with known effects of CORT on obligate metamorphosis in amphibians, our results link glucocorticoids to ontogenetic transitions in facultatively paedomorphic salamanders. We discuss our results in the context of theoretical models and the suite of environmental cues known to influence facultative paedomorphosis.

Towards comparative analyses of salamander limb regeneration.

Among tetrapods, only salamanders can regenerate their limbs and tails throughout life. This amazing regenerative ability has attracted the attention of scientists for hundreds of years. Now that large, salamander genomes are beginning to be sequenced for the first time, omics tools and approaches can be used to integrate new perspectives into the study of tissue regeneration. Here we argue the need to move beyond the primary salamander models to investigate regeneration in other species. Salamanders at first glance come across as a phylogenetically conservative group that has not diverged greatly from their ancestors. While salamanders do present ancestral characteristics of basal tetrapods, including the ability to regenerate limbs, data from fossils and data from studies that have tested for species differences suggest there may be considerable variation in how salamanders develop and regenerate their limbs. We review the case for expanded studies of salamander tissue regeneration and identify questions and approaches that are most likely to reveal commonalities and differences in regeneration among species. We also address challenges that confront such an initiative, some of which are regulatory and not scientific. The time is right to gain evolutionary perspective about mechanisms of tissue regeneration from comparative studies of salamander species.

Immunity in salamander regeneration: Where are we standing and where are we headed?

Salamanders exhibit the most extensive regenerative repertoire among vertebrates, being able to accomplish scar-free healing and faithful regeneration of significant parts of the eye, heart, brain, spinal cord, jaws and gills, as well as entire appendages throughout life. The cellular and molecular mechanisms underlying salamander regeneration are currently under extensive examination, with the hope of identifying the key drivers in each context, understanding interspecies differences in regenerative capacity, and harnessing this knowledge in therapeutic settings. The immune system has recently emerged as a potentially critical player in regenerative responses. Components of both innate and adaptive immunity have been found at critical stages of regeneration in a range of salamander tissues. Moreover, functional studies have identified a requirement for macrophages during heart and limb regeneration. However, our knowledge of salamander immunity remains scarce, and a thorough definition of the precise roles played by its members is lacking. Here, we examine the evidence supporting roles for immunity in various salamander regeneration models. We pinpoint observations that need revisiting through modern genetic approaches, uncover knowledge gaps, and highlight insights from various model organisms that could guide future explorations toward an understanding of the functions of immunity in regeneration.

Characterizing the regenerative capacity and growth patterns of the Texas blind salamander (Eurycea rathbuni).

BACKGROUND: Regeneration of complex patterned structures is well described among, although limited to a small sampling of, amphibians. This limitation impedes our understanding of the full range of regenerative competencies within this class of vertebrates, according to phylogeny, developmental life stage, and age. To broaden the phylogenetic breath of this research, we characterized the regenerative capacity of the Texas blind salamander (Eurycea rathbuni), a protected salamander native to the Edwards Aquifer of San Marcos, Texas and colonized by the San Marcos Aquatic Resource Center. As field observations suggested regenerative abilities in this population, the forelimb stump of a live captured female was amputated in the hopes of restoring the structure, and thus locomotion in the animal. Tails were clipped from two males to additionally document tail regeneration. RESULTS: We show that the Texas blind salamander exhibits robust limb and tail regeneration, like all other studied Plethodontidae. Regeneration in this species is associated with wound epithelium formation, blastema formation, and subsequent patterning and differentiation of the regenerate. CONCLUSIONS: The study has shown that the Texas blind salamander is a valuable model to study regenerative processes, and that therapeutic surgeries offer a valuable means to help maintain and conserve this vulnerable species.

Salamander-Eci: An optical clearing protocol for the three-dimensional exploration of regeneration.

BACKGROUND: Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms. RESULTS: Here we introduce Salamander-Eci, a rapid and robust optical clearing protocol optimized for the widely used axolotl model, which allows simultaneous immunohistochemistry and Click-chemistry detection with minimal volume disruption. We provide examples of its application, from whole larva to adult limbs and organs, and complement it with an image analysis pipeline for volumetric cell quantification. Further, we offer a detailed 3D quantitation of cell proliferation throughout axolotl limb regeneration. CONCLUSIONS: Salamander-Eci enables the comprehensive volumetric analysis of regenerative phenomena at both local and systemic levels.