Bufo Gargarizans Research Articles

Retinal ganglion cell distribution and spatial resolution in the Asiatic toad Bufo gargarizans (Günther, 1859)

Vision plays a crucial role in the biology of anurans. The spatial arrangement of retinal ganglion cells (GCs) is closely related to visual behavior in vertebrates. There is scarce data on GC topography in anurans, in particular, in toads. I studied the number and distribution of GCs in the retina of the Asiatic toad Bufo gargarizans. GCs were unevenly distributed across the retina. Their spatial density was minimum in the dorsal periphery (3374 and 2486 cells/mm2 in the smaller and larger toad, respectively). It increased towards the retinal equator, where a moderately pronounced visual streak was observed comprising several “patches” of a greater GC density. The streak had somewhat “vague” dorsal and ventral borders. The maximum GC density (8605 and 7282 cells/mm2 in the smaller and larger toad, respectively) was found in the temporal retina, slightly dorsal to the equator. The respective zone was identified as an area centralis. The total GC number ranged from 266 × 103 (smaller toad) to 309 × 103 cells (larger toad). The spatial resolution as estimated from eye geometry and GC density in air was minimum in the dorsal periphery (0.90 and 0.79 cycles per degree in smaller and larger toads, respectively) and maximum in the area centralis (1.43 and 1.36 cycles per degree in smaller and larger toads, respectively). Both retinal specializations found in the Asiatic toad match its biology.

Cell affinity screening combined with nanoLC-MS/MS based peptidomics for identifying cancer cell binding peptides from Bufo Bufo gargarizans

Animal venoms contain many peptides with high specificity and selectivity against their protein targets, a characteristic which makes venoms an invaluable source of potential drugs. High-sensitivity mass spectrometry (MS)- based peptidomic platform has evolved as a predominant method for natural peptide drug discovery due to its strength for direct and rapid identification of peptides and peptide-associated post-translational modifications (PTMs). In this study, we used cell-affinity assays combined with nanoLC-MS/MS based peptidomics to identify cancer cell binding peptides (CBPs) from Bufo Bufo gargarizans. We identified 76 potential cell binding peptides and 237 non-affinity peptides in venom extracts from Asiatic toads, and some were verified with MS-parallel reaction monitoring (PRM) mode. These peptides were further analyzed and internalized within human cells and some demonstrated anti-tumor properties in vitro. These specific peptides might be used as templates for peptide-based drug design or optimization.

Morphology and mechanical performance between the skin surface ofRana dybowskiiandBufo gargarizans

Anuran skin is a typical natural biomaterial with multifunctional features. A specific comparison of mechanical performance and morphology related to them was performed in the skin of Rana dybowskii and Bufo gargarizans using the tensile testing technique and morphological equipment. Rana dybowskii's skin has soft smooth surface covered by polygonal epidermal cells, while the Bufo gargarizans species has tough and uneven skin surface due to numerous verrucae structures. The collagen fibre bundles in lower dermis of Bufo gargarizans have wavelike organisation while the bundles of Rana dybowskii show a parallel arrangement. The mean elastic modulus of Rana dybowskii was nine times higher than that of Bufo gargarizans. This study clarified that the arrangement of collagen fibres play an important role in the strength and elasticity of skin material.

Decisive Effects of Life Stage on the Gut Microbiota Discrepancy Between Two Wild Populations of Hibernating Asiatic Toads (Bufo gargarizans).

Until now, the effects of driving factors on the gut microbiota of amphibians are still mostly confounded. Due to a long-term fasting, hibernating amphibians are ideal experimental materials to explore this question. In this study, we characterized the small intestine microbiota of adult hibernating Asiatic toads (Bufo gargarizans) collected from two geographical populations using 16S rRNA amplicon sequencing technique and evaluated the effects of non-dietary factors (e.g., sex and host genetic background). Proteobacteria (0.9196 ± 0.0892) was characterized as the most dominant phylum in the small gut microbiota of hibernating Asiatic toads, among which five core OTUs were identified and three were classified into Pseudomonas. In view of the coincidence between the dominant KEGG pathways (such as the two-component system) and Pseudomonas, Pseudomonas appeared to be a key adaptor for small gut microbiota during hibernation. Furthermore, we detected a greater discrepancy of gut microbiota between geographical populations than between sexes. Both sex and host genetic background showed a minor effect on the gut microbiota variation. Finally, life stage was determined to be the decisive factor driving the gut microbiota discrepancy between populations. However, a large proportion of the gut microbiota variation (∼70%) could not be explained by the measured deterministic factors (i.e., sex, location, body length, and routine blood indices). Therefore, other factors and/or stochastic processes may play key roles in shaping gut bacterial community of hibernating amphibians.

Investigation of cytotoxic effect of the bufanolide steroid compound cinobufagin and its related underlying mechanism in brain cell models.

Cinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well asan analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN-2 neuronal cell line. In GHA cells but not in HCN-2 cells, cinobufagin (20-60 μM) induced [Ca2+ ]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid reduced cinobufagin-induced cytotoxicity in GHA cells. In GHA cells, cinobufagin-induced Ca2+ entry was inhibited by 2-aminoethoxydiphenyl borate or SKF96365. In a Ca2+ -free medium, treatment with thapsigargin or U73122 abolished cinobufagin-evoked [Ca2+ ]i rises. Furthermore, treatment with N-acetylcysteine reversed ROS production and cytotoxicity in cinobufagin-treated GHA cells. Together, in GHA cells but not in HCN-2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+ ]i rises by Ca2+ influx via store-operated Ca2+ entry and phospholipase C-dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS-associated cytotoxicity.

Inhibition of Metamorphosis, Thyroid Gland, and Skeletal Ossification Induced by Hexavalent Chromium in Bufo gargarizans Larvae.

Hexavalent chromium (Cr [VI]) is one of the major detrimental heavy metal pollutants. In the present study, Bufo gargarizans were exposed to 0, 52, 104, 208, and 416 μg/L Cr (VI) from Gosner stage 2 until metamorphosis; and growth, development, and histological characteristics of the thyroid gland and skeletal ossification were examined. The results demonstrated that the survival rate of larvae exposed to Cr (VI) was not different from that measured in animals from the control group. However, high levels of Cr (VI) (104, 208, and 416 μg/L) were associated with significantly delayed growth and development. The suppression of skeletal ossification was observed at high Cr (VI) levels. Besides, histological alterations of the thyroid gland, such as follicular cell hyperplasia, colloid depletion, and peripheral colloid vacuolation, were found in 52 to 416 μg/L Cr (VI) treatments. The results of the present study highlight reductions in growth and development as well as percent metamorphosis and skeletal ossification due to histological alteration of the thyroid gland during exposure to Cr (VI) in B. gargarizans larvae. The present investigation could provide a basis for understanding the detrimental effects of Cr (VI) in amphibian larvae. Environ Toxicol Chem 2021;40:2474-2483. © 2021 SETAC.

Altered temperature affect body condition and endochondral ossification in Bufo gargarizans tadpoles.

Bufo gargarizans is one kind of economic animals with higher medicinal value in China. In this study, B. gargarizans (Bufo gargarizans) tadpoles were reared at three different water temperature (15, 22 and 29°C) from Gosner stages 28-46. We investigated the effects of temperature on growth, development, survival, metamorphic duration, size and skeletal ossification at Gosner stage 40, 42, and 46, as well as thyroid tissue reached metamorphic climax (Gs42). Besides, we examined the transcription levels of endochondral ossification-related genes in hind limb at metamorphic climax (Gs42). Our results showed that the growth and development of tadpoles conform to the temperature-size rule (TSR). While warm temperature resulted in the decrease in body size and hind limb length, and shorten larval period, cold temperature led to increase in body size and hind limb length but prolonged larval period. Histological examinations revealed that warm and cold temperatures caused damage to thyroid tissue. Also, warm and cold temperatures inhibited the degree of ossification with the double staining methodology. Additionally, the real-time PCR results suggested that warm and cold temperatures significantly up-regulated Runx2, VEGF and VEGFR mRNA levels, and down-regulated TRβ, MMP9, MMP13 and Runx3 mRNA levels. The up-regulation of Dio2 level and down-regulation of Dio3 level were observed in warm temperature. TRα mRNA level was significantly increased in warm temperature, but decreased in cold temperature. Collectively, these observations demonstrated that warm and cold temperatures affected endochondral ossification in B. gargarizans tadpoles, which might influence their capacity to terrestrial locomotion.

Effects of microhabitat characteristics on amphibian diversity in urban water system of Kaifeng, Henan, China.

Amphibians with a unique life history are extremely sensitive to environmental changes. They are important indicator species for environmental change. The construction of Kaifeng water system affects the habitat and biodiversity of amphibians. In this study, we investigated the amphi-bians in water system of Kaifeng by setting sample points and using visual encounter method, and measured the habitat variables. We quantified amphibian biodiversity under different habitat types using the Shannon diversity index, Pielou evenness index and Simpson dominance index, and explored the responses of amphibians to microhabitat variations by cluster analysis and redundancy analysis. The results showed that the diversity, evenness, and dominance of amphibians in the natural revetment were higher than those in the artificial hardened revetment, indicating a more stable amphibian population in the natural habitat. The dominance index of amphibians on natural revetment was higher than that of the two artificially hardened revetments, indicating that amphibians preferred natural habitat. The abundance of both Palophylax nigromaculata and Bufo gargarizans had significant positive correlation with night light intensity, while the abundance of P. plancyi was positively correlated with total phosphorus, and that of Fejervarya limnocharis was positively correlated with water pH. The protection of amphibians should be considered in the construction of urban water system in Kaifeng. The presence of more natural revetment would be of great significance to enhance amphibian biodiversity.

Processing evolution,chemical components and pharmacological effects of Bufonis Venenum

Bufonis Venenum,the dried secretion of Bufo bufo gargarizans or B. melanostictus,is toxic and hard with the efficacy of removing toxicity for detumescence and relieving pain. The processing of Bufonis Venenum dates back to the Song dynasty. In addition to the wine-processing,milk-processing and talcum powder-processing,there were some other kinds of processing methods in ancient times,such as baking,calcining,water-soaking and vinegar-processing. Modern studies have shown that the Bufonis Venenum has the main chemical components of bufadienolides,indole alkaloids sterols,and other compounds. It has the pharmacological effects of antitumor,cardiac,antibacterial,and analgesic activities,local anesthesia,and so on. This paper reviews the processing evolution,chemical components and pharmacological effects of Bufonis Venenum,providing references for its special processing and modern research as well as the theoretical basis for the research on its processing mechanism and quality control.

Metabolic cold adaptation in the Asiatic toad: intraspecific comparison along an altitudinal gradient.

The metabolic cold adaptation (MCA) hypothesis predicts an increase in metabolic rate and thermal sensitivity of poikilotherms from cold environments as compared to those from warm environments, when measured under standardized conditions. This compensatory response is also expected to evolve in life history and behavioral traits if the reductions in these phenotypic traits at low temperature involves in a reduction in fitness. We investigated the extent to which the level of energy intake (measured as feeding rate), energy turnover (measured as standard metabolic rate, SMR) and the energy budget (energy allocation to growth and physical activity) are influenced by climatic conditions in three populations of the Asiatic toad (Bufo gargarizans) distributed across an altitudinal gradient of 1350m in the Qionglai Mountains of Western China. We found a similar thermal reaction norm of SMR at both population and individual levels; therefore, the data did not support the MCA hypothesis. However, there was a co-gradient variation (CoGV) for mass change rate in which the high and medium altitudinal populations displayed slower mass change rates than their counterparts from low altitudes. Moreover, this CoGV pattern was accompanied by a low feeding rate and high physical activity for the high- and medium-altitude populations. Our results highlight that adjustments in energy intake and energy allocation to behaviors, but not energy allocation to metabolism of maintenance, could act as an energetic strategy to accommodate the varied growth efficiency in Asiatic toads along an altitudinal gradient.

Pseudogenized Amelogenin Reveals Early Tooth Loss in True Toads (Anura: Bufonidae).

Extant anurans (frogs and toads) exhibit reduced dentition, ranging from a lack of mandibular teeth to complete edentulation, as observed in the true toads of the family Bufonidae. The evolutionary time line of these reductions remains vague due to a poor fossil record. Previous studies have demonstrated an association between the lack of teeth in edentulous vertebrates and the pseudogenization of the major tooth enamel gene amelogenin (AMEL) through accumulation of deleterious mutations and the disruption of its coding sequence. In this study, we have harnessed the pseudogenization of AMEL as a molecular dating tool to correlate loss of dentition with genomic mutation patterns during the rise of the family Bufonidae. Specifically, we have utilized AMEL pseudogenes in three members of the family as a tool to estimate the putative date of edentulation in true toads. Comparison of AMEL sequences from Rhinella marina, Bufo gargarizans and Bufo bufo, with nine extant, dentulous frogs, revealed mutations confirming AMEL inactivation in Bufonidae. AMEL pseudogenes in modern bufonids also exhibited remarkably high 86-93% sequence identity among each other, with only a slight increase in substitution rate and relaxation of selective pressure, in comparison with functional copies in other anurans. Moreover, using selection intensity estimates and synonymous substitution rates, analysis of functional and pseudogenized AMEL resulted in an estimated inactivation window of 46-60 million years ago in the lineage leading to modern true toads, a time line that coincides with the rise of the family Bufonidae.

First distribution record of the Asiatic Toad Bufo gargarizans Cantor, 1842 from India — Dibang Valley in Arunachal Pradesh

Bufo gargarizans, a species complex, has a wide distribution ranging from Japan to south-western China, Vietnam, and Russia but was not previously reported from India. Surveys conducted in Dibang Valley district of Arunachal Pradesh near the Indo-Tibetan border with China in 2014–15 revealed previously unreported specimens of the toad genus Bufo. Based on photographic comparisons with morphological characteristics obtained from published literature, we have identified the Bufo from Dibang Valley as the Asiatic Toad Bufo gargarizans Cantor, 1842. Individuals of the species reported from Dibang have a wide mid dorsal line in the dorsum, rarely observed in Bufo gargarizans except in the sub-populations on the Tibetan Plateau.

Bufadienolides from the Eggs of the Toad Bufo bufo gargarizans and Their Antimelanoma Activities.

Toads produce potent toxins, named bufadienolides, to defend against their predators. Pharmacological research has revealed that bufadienolides are potential anticancer drugs. In this research, we reported nine bufadienolides from the eggs of the toad Bufo bufo gargarizans, including two new compounds (1 and 3). The chemical structures of 1 and 3, as well as of one previously reported semisynthesized compound (2), were elucidated on the basis of extensive spectroscopic data interpretation, chemical methods, and X-ray diffraction analysis. Compound 1 is an unusual 19-norbufadienolide with rearranged A/B rings. A biological test revealed that compounds 2 and 4-8 showed potent cytotoxic activities toward human melanoma cell line SK-MEL-1 with IC50 values less than 1.0 μM. A preliminary mechanism investigation revealed that the most potent compound, 8, could induce apoptosis via PARP cleavage, while 5 and 6 significantly suppressed angiogenesis in zebrafish. Furthermore, an in vivo biological study showed that 5, 6, and 8 inhibit SK-MEL-1 cell growth significantly.

Comparison of hematological traits and oxygenation properties of hemoglobins from highland and lowland Asiatic toad (Bufo gargarizans).

The Asiatic toad (Bufo gargarizans) belonging to the family of Bufonidae (Anura: Amphibia) is successfully residing on the Qinghai-Tibetan Plateau (QTP). To investigate whether the oxygen delivery undergoes adaptive adjustments to high-altitude environments in Asian toads inhabiting the QTP (Zoige County, 3446m), choosing low-altitude populations (Chengdu City, 500m) as control, we measured hematological traits, O2 affinities of whole blood, Hb-O2 affinities of purified Hbs, their sensitivities to temperature, and allosteric effectors (H+, Cl- and ATP). Our results showed that high-altitude Asiatic toads possessed significantly increased hemoglobin concentration, hematocrit, and red blood cell count, but significantly decreased erythrocyte volume compared with low-altitude toads. The whole blood and purified Hbs of high-altitude Asiatic toads both exhibited significantly higher O2 affinities compared with low-altitude toads. Substantially increased intrinsic Hb-O2 affinities of high-altitude Asiatic toads Hbs are likely to be the main reason for its elevated Hb-O2 affinities given the anionic cofactor sensitivities of high- and low-altitude toads were similar. The Hbs of high-altitude toads were also characterized by distinctly strong Bohr effects at the low temperature and low-temperature sensitivities. The adaptive adjustments of hematological traits could enhance the blood-O2 carrying capacity of high-altitude Asiatic toads. The increased Hb-O2 affinities could safeguard the pulmonary O2 uploading under hypoxia. The strong Bohr effects at the low temperature could help the release of O2 in metabolic tissues and cold limbs, while low-temperature sensitivity could minimize the effect of temperature fluctuation on the Hb-O2 affinity.

Copper-induced sublethal effects in Bufo gargarizans tadpoles: growth, intestinal histology and microbial alternations.

Copper (Cu) is one of the environmental contaminations which can pose significant risks for organisms. The current study explores the effects of Cu exposure on the growth, intestinal histology and microbial ecology in Bufo gargarizans. The results revealed that 0.5-1 μM Cu exposure induced growth retardation (including reduction of total body length and wet weight) and intestinal histological injury (including disordered enterocyte, changes in the villi and vacuoles) of tadpoles. Also, high-throughput sequencing analysis showed that Cu exposure caused changes in richness, diversity and structure of intestinal microbiota. Moreover, the composition of intestinal microbiota was altered in tadpoles exposed to different concentrations of Cu. At the phylum level, we observed the abundance of proteobacteria was increased, while the abundance of fusobacteria was decreased in the intestinal microbiota of tadpoles exposed to 1 μM Cu. At the genus level, a reduced abundance of kluyvera and aeromonas was observed in the intestinal microbiota of tadpoles under the exposure of 0-0.5 μM Cu. Finally, functional predictions revealed that tadpoles exposed to copper may be at a higher risk of developing metabolic disorders or diseases. Above all, our results will develop a comprehensive view of the Cu exposure in amphibians and will yield a new consideration for sublethal effects of Cu on aquatic organisms.

A large genome with chromosome-scale assembly sheds light on the evolutionary success of a true toad (Bufo gargarizans).

We present a high-quality genome assembly for the Asiatic toad (Bufo gargarizans) and explore the evolution of several large gene families in amphibians. With a large genome assembly size of 4.55Gb, the chromosome-scale assembly includes 747 scaffolds with an N50 of 539.8Mb and 1.79% gaps. Long terminal repeats (LTRs) constitute a high proportion of the genome and their expansion is a key contributor to the inflated genome size in this species. This is very different from other small amphibian genomes, but similar to that of the enormous axolotl genome. The genome retains a large number of duplicated genes, with tandem (TD) and proximal duplications (PD) the predominant mode of duplication. A total of 122 gene families have undergone significant expansion and were mainly enriched in sensory perception of smell and bitter taste. The CYP2C subfamily, which plays an important role in metabolic detoxification, specifically expanded via TD and PD in the Asiatic toad and the cane toad (true toads). Most of Na+ /K+ -ATPase genes experienced accelerated evolution along Bufonid lineages and two amino acid sites involving toad-toxin resistance were found to experience positive selection. We also revealed a dynamic evolution of olfactory and vomeronasal receptor gene families which was likely driven by the water-to-land transition. The high-quality genome of the Asiatic toad will provide a solid foundation to understand the genetic basis of its many biological processes.

The Effectiveness and Safety of Cinobufotalin Injection as an Adjunctive Treatment for Lung Cancer: A Meta-Analysis of Randomized Controlled Trials.

Cinobufotalin injection is a water-soluble preparation extracted from the skin secretion of Bufo bufo gargarizans Cantor or B. melanotictus Schneider, which has been widely used as an adjuvant treatment in lung cancer patients. This study aimed to evaluate the clinical efficacy and safety of cinobufotalin (PubChem CID: 259776) injection as an adjunctive treatment for lung cancer. We designed a meta-analysis that performed following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We aim to include randomized controlled trials by systematically searching the PubMed, EMBASE, CNKI, Wanfang database, VIP, CBM, the Cochrane Central Register of Controlled Trials, and Chinese Clinical Trial Registry from inception to Mar 1, 2020, comparing the difference between the use of cinobufotalin injection as an adjunctive treatment and a control group without cinobufotalin injection. The objective response rate (ORR) and quality of life (QOL) will be defined as the primary outcomes, and the disease control rate (DCR) and adverse events will be defined as the secondary outcomes. We included 21 articles with 1735 cases of lung cancer patients. Comparison results show that combining with cinobufotalin injection can improve ORR (OR = 1.77, 95% CI [1.43, 2.21], P < 0.001), with low heterogeneity (P = 0.94, I2 = 0%); DCR (OR = 2.20, 95% CI [1.70, 2.85], P < 0.001), with low heterogeneity (P = 0.60, I2 = 0%); KPS score (OR = 3.10, 95% CI [2.23, 4.32], P < 0.001), with low heterogeneity (P = 0.85, I2 = 0%); and the effect of pain relief (OR = 2.68, 95% CI [1.30, 5.55], P = 0.008), with low heterogeneity (P = 0.72, I2 = 0%). Low-to-moderate evidence shows that cinobufotalin injection combined with chemotherapy can significantly increase ORR, DCR, QOL, and the effect of pain relief. Meanwhile, cinobufotalin injection did not bring additional adverse events such as hematological toxicity, gastrointestinal toxicity, cardiotoxicity, hepatotoxicity, and nephrotoxicity; however, multicenter, large-sample, high-quality clinical research results are still needed to reveal the therapeutic effect of cinobufotalin injection in small-cell lung cancer (PROSPERO registration number: CRD42020170052).

Physiological, developmental, and behavioral plasticity in response to thermal acclimation

Organisms with complex ecologies and life-cycle processes may shift physiologically (acclimation in tolerance), developmentally, and/or behaviorally (thermoregulation) in response to changes in climate. As such, climate change may trigger multiple, interacting phenotypic responses, which underscores the nuances of characterizing a species capacity to adapt and respond to climate change. In this study, we use a model frog species, Bufo gargarizans, to examine how three phenotypes, thermal tolerance limits (critical thermal minimum, CTmin and critical thermal maximum, CTmax), ontogeny, and behavioral preferences in temperature (Tpref) respond to different levels of thermal exposure (i.e., acclimation ranging from 10°C to 30°C). Acclimation temperature had little effect on Tpref of tadpoles, yet behaviorally they showed strong signs of thermal selection towards an optimum. Both CTmin and CTmax increased with acclimation temperature with an approximate 10% increase in tolerance limits per 1°C increase in exposure. Development and body size both responded to acclimation temperature, both of which also influenced lower but not upper thermal limits. Our study highlights the idiosyncrasies of estimating climate vulnerability, where multiple phenotypes can respond to shifts in temperature-a complexity that is especially apparent in species with complex life-cycles.

Cloning and characterization of steroid 5β-reductase from the venom gland of Bufo bufo gargarizans

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, “cholesterol-bile acid-bufadienolidies pathway” was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3β-hydroxysteroid dehydrogenase (3βHSD) and steroid 5β-reductase (SRD5β) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5β cDNA of B. bufo gargarizans (Bbg-SRD5β) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5β. Bbg-SRD5β had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5β in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5β was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5β can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5β-Y132G was 1.8 times higher than that of wild type Bbg-SRD5β. Although Bbg-SRD5β was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5β is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5β.

Brain size variation along altitudinal gradients in the Asiatic Toad (Bufo gargarizans).

Size changes in brain and brain regions along altitudinal gradients provide insight into the trade‐off between energetic expenditure and cognitive capacity. We investigated the brain size variations of the Asiatic Toad (Bufo gargarizans) across altitudes from 700 m to 3,200 m. A total of 325 individuals from 11 sites and two transects were sampled. To reduce confounding factors, all sampling sites within each transect were within a maximum distance of 85 km and an altitudinal difference close to 2,000 m. Brains were dissected, and five regions were both measured directly and with 3D CT scan. There is a significant negative correlation between the relative whole‐brain volume (to snout‐vent length) and altitude. Furthermore, the relative volumes (to whole‐brain volume) of optic tectum and cerebellum also decrease along the altitudinal gradients, while the telencephalon increases its relative volume along the gradients. Therefore, our results are mostly consistent with the expensive brain hypothesis and the functional constraint hypothesis. We suggest that most current hypotheses are not mutually exclusive and data supporting one hypothesis are often partially consistent with others. More studies on mechanisms are needed to explain the brain size evolution in natural populations.