Spatio-temporal analysis of toxigenic genes expression in the growing Bufo gargarizans based on RNA sequencing data.

BACKGROUND: Bufo gargarizans Cantor, a widely distributed amphibian species in Asia, produces and releases toxins through its retroauricular and granular glands. Although various tissues have been sequenced, the molecular mechanisms underlying the toxin production remain unclear. To elucidate these mechanisms, abdominal skin (non-toxic secretory glands) and retroauricular gland (toxic secreting glands) samples were collected at different time points (3, 6, 12, 24, and 36 months) for RNA sequencing (RNA-seq) and analysis. RESULTS: In comparison to the S group during the same period, a total of 3053, 3026, 1516, 1028, and 2061 differentially expressed genes (DEGs) were identified across five developmental stages. Gene Ontology (GO) analysis revealed that DEGs were primarily enriched in biological processes including cellular processes, single-organism processes, metabolic processes, and biological regulation. In terms of cellular components, the DEGs were predominantly localized in the cell and cell parts, whereas molecular function indicated significant enrichment in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the metabolism and synthesis of various substances, such as lipid metabolism, cofactor and vitamin metabolism, tryptophan metabolism, steroid biosynthesis, and primary bile acid biosynthesis, were accompanied by the development of toads. Additionally, using trend analysis, we discovered candidate genes that were upregulated in the retroauricular glands during development, and the abundance of these genes in the abdominal skin was extremely low. Finally, we identified 26 genes that are likely to be involved in toxin production and that are likely to be involved in toxin anabolism. CONCLUSION: Overall, these results provide new insights into the genes involved in toxin production in B. gargarizans, which will improve our understanding of the molecular mechanisms underlying toxigenic gene expression.

Thermal conditions predict intraspecific variation in senescence rate in frogs and toads.

Variation in temperature is known to influence mortality patterns in ectotherms. Even though a few experimental studies on model organisms have reported a positive relationship between temperature and actuarial senescence (i.e., the increase in mortality risk with age), how variation in climate influences the senescence rate across the range of a species is still poorly understood in free-ranging animals. We filled this knowledge gap by investigating the relationships linking senescence rate, adult lifespan, and climatic conditions using long-term capture-recapture data from multiple amphibian populations. We considered two pairs of related anuran species from the Ranidae (Rana luteiventris and Rana temporaria) and Bufonidae (Anaxyrus boreas and Bufo bufo) families, which diverged more than 100 Mya and are broadly distributed in North America and Europe. Senescence rates were positively associated with mean annual temperature in all species. In addition, lifespan was negatively correlated with mean annual temperature in all species except A. boreas In both R. luteiventris and A. boreas, mean annual precipitation and human environmental footprint both had negligible effects on senescence rates or lifespans. Overall, our findings demonstrate the critical influence of thermal conditions on mortality patterns across anuran species from temperate regions. In the current context of further global temperature increases predicted by Intergovernmental Panel on Climate Change scenarios, a widespread acceleration of aging in amphibians is expected to occur in the decades to come, which might threaten even more seriously the viability of populations and exacerbate global decline.

Mining and Characterization of Indolethylamine N-Methyltransferases in Amphibian Toad Bufo gargarizans.

Strong, psychedelic indolethylamines (IAAs) are typically present in trace amounts in the majority of species, but they build up significantly in the skin of amphibian toads, especially N-methylated 5-hydroxytryptamine (5-HT) analogues. However, there is no pertinent research on the investigation of indoleamine N-methyltransferase (INMT) in amphibians, nor is there any adequate information on the key amino acids that influence the activity of known INMTs from other species. Herein, we focused on Bufo toad INMT (BINMT) for the first time and preliminarily identified BINMT 1 from the transcriptomes of Bufo gargarizans active on tryptamine, 5-HT, and N-methyl-5-HT. We established the enzyme kinetic characteristics of BINMT 1 and identified the essential amino acids influencing its activity via molecular docking and site-directed mutagenesis. Subsequently, we carried out sequence alignment and phylogenetic tree analysis on 43 homologous proteins found in the genome of B. gargarizans with BINMT 1 as the probe and selected seven of them for protein expression and activity assays. It was found that only three proteins possessing the highest similarity to BINMT 1 had INMT activity. Our research unveils the binding residues of BINMT for 5-HT analogues for the first time and initiates the study of INMTs in amphibian toads, serving as a tentative reference for further study of BINMT and providing insight into the comprehension of BINMT's catalytic mechanism and its role in the biosynthesis of 5-HT analogues in Bufo toads. It also contributes to the expansion of the INMT library to help explore and explain interspecies evolution in the future.

Metabolic Adaption of Flexor Carpi Radialis to Amplexus Behavior in Asiatic Toads (Bufo gargarizans).

Amplexus is a type of mating behavior among toads that is essential for successful external fertilization. Most studies have primarily focused on the behavioral diversity of amplexus, and less is known regarding the metabolic changes occurring in amplectant males. The aim of this study was to compare the metabolic profiles of amplectant Asiatic toad (Bufo gargarizans) males in the breeding period (BP group) and the resting males in the non-breeding period (NP group). A metabolomic analysis was conducted on the flexor carpi radialis (FCR), an essential forelimb muscle responsible for clasping during courtship. A total of 66 differential metabolites were identified between the BP and NP groups, including 18 amino acids, 12 carbohydrates, and 8 lipids, and they were classified into 9 categories. Among these differential metabolites, 13 amino acids, 11 carbohydrates, and 7 lipids were significantly upregulated in the BP group compared to the NP group. In addition, a KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis identified 17 significant metabolic pathways, including ABC transporters, aminoacyl-tRNA biosynthesis, arginine biosynthesis, pantothenate and CoA biosynthesis, and fructose and mannose metabolism. These results suggest that amplectant male toads are metabolically more active than those during the non-breeding period, and this metabolic adaptation increases the likelihood of reproductive success.

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

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

Effects of temperature on growth, development and the leptin signaling pathway of Bufo gargarizans.

Climate change is one of the most important causes of the decline in amphibians. Changes in temperature have an important effect on the growth and development and energy metabolism of amphibians. The aim of this study is to unravel the effects of temperature on the leptin signaling pathway of Bufo gargarizans and its molecular mechanisms. Our results showed that high temperature accelerated the development rate of tadpoles, but reduced body size and mass, while low temperature deferred the development of tadpoles, but increased size and mass. Both high temperature and low temperature exposure caused pathological damage of the liver in B. gargarizans. The results of RT-qPCR revealed that the high temperature treatment significantly upregulated the transcript levels of genes related to thyroid hormone (DIO2 (D2), Thyroid Hormone Receptor-α (TRα)) and the leptin signaling pathway (Leptin Receptor (LepR), Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Tyrosine kinase 2 (TYK2), Signal Transducer And Activator Of Transcription 3 (STAT3), Signal Transducer And Activator Of Transcription 3.1 (STAT3.1), and Signal Transducer And Activator Of Transcription 6 (STAT6)), while there was a decrease of mRNA expression of these genes (TRα, Thyroid Hormone Receptor-Beta (TRß), LepR, JAK1, and TYK2) in the liver of tadpoles exposed to high temperature compared with the intermediate temperature treatment. Therefore, our results suggested that temperature extremes might interfere with the thyroid and leptin signaling pathways and affect the growth and development of B. gargarizans. Furthermore, tissue injury of the liver could occur due to exposure to temperature extremes. This work promotes public awareness of environmental protection and species conservation needs, also provides valuable experimental data and a theoretical basis for the protection of amphibians.

Bufadienolides from the Skin Secretions of the Neotropical Toad Rhinella alata (Anura: Bufonidae): Antiprotozoal Activity against Trypanosoma cruzi.

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16ß-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16ß-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16ß-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1-10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.

Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism.

(1) Background: Toad venom (Bufonis Venenum, known as 'Chansu' in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.

Response Patterns of Biomarkers as Tools to Identify Toxic Effects of Cadmium and Lead on Bufo gargarizans Embryo.

Molecular biomarkers play an increasing crucial role in evaluating and predicting toxicity of metals. Expressions patterns of genes related to oxidative stress, apoptosis, immune and inflammation response in the Bufo gargarizans embryo exhibited a development dependent manner. The genes related to oxidative stress (HSP, GPx and SOD) are the first response in the development of embryo, followed by the apoptosis (Bax, BCLAF1 and TRAIL) and inflammation and immune response (SOCS3, IL-27 and IL-17D), respectively. Then, we have verified the HSP, Bax and SOCS3 IL-27 (expressed highest in their respective processes) exhibited the most significant changes in Cd-Pb mixed group compared with control. In addition, we found exposure of Cd-Pb mixed metals causes greater adverse effects than Cd, Pb alone on development and morphology of embryo. Overall, our results provide a useful tool to use the sensitive molecular biomarkers as indicators of developmental toxicity in amphibian embryo.

The Mating Call of the Terrestrial Toad, Rhinella arenarum, as a Cue for Spatial Orientation and Its Associated Brain Activity.

Acoustic communication is essential for reproduction and predator avoidance in many anuran species. For example, mating calls are generally produced by males and represent a conspicuous communication signal employed during the breeding season. Although anuran mating calls have been largely studied to analyze content and phonotaxis toward choruses, they are rarely discussed as sources of information guiding spatial behavior in broader contexts. This is striking if we consider that previous studies have shown anurans to be impressive navigators. In the current study, we investigated whether terrestrial toad (Rhinella arenarum) males can use a mating call as a spatial cue to locate a water reward in a laboratory maze. Male toads could indeed learn the location of a reward guided by a mating call. This navigational ability, as indicated by c-Fos, was associated with greater neuronal activity in the telencephalic hippocampal formation (HF; also referred to in amphibians as medial pallium), the medial septum (MS), and the central amygdala (CeA). HF and MS are telencephalic structures associated with spatial navigation in mammals and other vertebrates. The CeA, by contrast, has been studied in the context of acoustic processing and communication in other amphibian species. The results are discussed in the framework of an evolutionary conserved, HF-septal spatial-cognitive network shared by amphibians and mammals.

Larval amphibians rapidly bioaccumulate poly- and perfluoroalkyl substances.

Poly- and perfluoroalkyl substances (PFAS) are ubiquitous contaminants that can bioaccumulate in aquatic taxa. Amphibians are particularly vulnerable to contaminants and sensitive to endocrine disruptors during their aquatic larval stage. However, few studies have explored PFAS uptake rates in amphibians, which is critical for designing ecotoxicology studies and assessing the potential for bioaccumulation. Uptake rates of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were measured for larval northern leopard frogs (Rana pipiens), American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum) during a 240-h exposure to 10 and 1000 µg/L concentrations. We measured body burden and calculated bioconcentration factor (BCF) every 48 h during the experiments. For all species and exposures, body burdens often reached steady state within 48-96 h of exposure. Steady-state body burdens for PFOA and PFOS ranged from 3819 to 16,481 ng/g dry weight (BCF = 0.46-2.5) and 6955-489,958 ng/g dry weight (47-259 BCFs), respectively. Therefore, PFAS steady state occurs rapidly in the larval amphibians we studied and particularly for PFOS. This result reflects a high potential for PFAS trophic transfer because amphibians are often low in trophic position and are important prey for many aquatic and terrestrial species.

The morphological changes and molecular biomarker responses in the liver of fluoride-exposed Bufo gargarizans larvae.

The goal of the current study was to evaluate the negative influences of fluoride on liver of Bufo gargarizans larvae. B. gargarizans larvae were treated with 42.4mgF-/L for 0, 24, 48 and 72h at Gosner stage 37. The morphological changes and responses of molecular biomarkers involved in lipid metabolism, oxidative stress and apoptosis were examined in liver. Disappearance of cell boundaries, degeneration of hepatic parenchyma cells and significant increase in the number of melanomacrophage centres and the quantity of lipid droplets were found in the liver treated with 42.4mgF-/L for 72h. In addition, in the relative expression of acetyl CoA carboxylase 1 (ACC-1), fatty acid elongase 1 (FAE-1), sterol carrier protein 2 (SCP-2), and carnitine palmitoyltransferase-1 (CPT-1), decrease was observed after 24, 48 and 72h of 42.4mgF-/L exposure. Furthermore, the transcript levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were downregulated in tadpoles exposed for 24, 48 and 72h to 42.4mgF-/L, while the transcript level of heat shock protein 90 (HSP90) was upregulated at 42.4mgF-/L for 72h. Also, mRNA expression of Bcl-2-associated transcription factor 1(BCLAF1) and thyroid hormone receptors (TRα and TRß) was significantly upregulated in tadpoles treated with 42.4mgF-/L for 72h. Therefore, our results suggested that the liver injury induced by fluoride might result from disruption of lipid metabolism, oxidative damage and apoptosis.

Comparative Analysis of Hydrophilic Ingredients in Toad Skin and Toad Venom Using the UHPLC-HR-MS/MS and UPLC-QqQ-MS/MS Methods Together with the Anti-Inflammatory Evaluation of Indolealkylamines.

Toad skin and toad venom, as two kinds of Chinese medicine, are prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. However, they display distinct properties in traditional application, and the hydrophilic ingredients are the possible distinguishing chemicals between them. In this work, 36 and 22 hydrophilic components were characterized from toad skin and venom, respectively, by UHPLC-HR-MS/MS, including amino acids, nucleosides, polypeptides, and indolealkylamines (IAAs). Among them, 15 compounds were unambiguously confirmed by comparison with standards. The CID-MS/MS fragmentation behaviors of seven indolealkylamine references were investigated to ascertain three types of structures. Subsequently, 11 high abundance contents of hydrophilic ingredients were determined from 11 batches of toad skin and 4 batches of toad venom by UPLC-QqQ-MS/MS. The quantitative results showed that the content of main IAAs in toad venom was much higher than in skin. In addition, the N-methyl serotonin (free IAA), bufothionine (combined IAA), and total IAAs sample were selected for anti-inflammatory evaluation in lipopolysaccharide (LPS) stimulated zebrafish embryo models. The obvious anti-inflammatory activities of IAAs were observed, especially for the free IAAs. This study illustrated IAAs were the main distinct hydrophilic components that probably lead to the difference between toad skin and toad venom in traditional applications.

Effects of Zn and Ti Nanoparticles on the Survival and Growth of Sclerophrys arabica Tadpoles in a Two Level Trophic System.

Under natural conditions pollutants rarely affect only a single trophic level. This study investigated the effects of titanium dioxide (Ti-NPs) and zinc oxide (Zn-NPs) nanoparticles on survival and growth of Sclerophrys arabica tadpoles exposed directly in water, indirectly through their food source (decomposing leaves), or a combination of both. Zn-NPs did not cause significant mortality. The LC50 for tadpoles exposed to Ti-NPs directly was 74.9 µg/L (95% CI 16.6-338.7 µg/L) and 18.3 µg/L (95% CI 6.5-51.9 µg/L) for tadpoles exposed directly and with pre-treated leaves. Tadpoles fed only pre-exposed food did not show significant mortality. Exposure route also affected growth. On average the tadpoles with the lowest body mass were found in the water-only exposures, followed by the water and leaf treatments. However, the tadpoles with the greatest body mass were in the treatments fed with leaves pretreated with Ti-NPs. This suggests that the NPs made their contents more available to the tadpoles.

Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen.

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides-the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

[Effects of different stocking density and diet on development of Bufo gargarizans tadpoles].

The effects of four different stocking densities and five different diets on the growth of Bufo gargarizans tadpoles were studied to determine the optimum stocking density and diet. For stocking density experiment, the tadpoles were fed respectively at different density of 200, 500, 1 000 and 2 000 tadpoles per square meter. For diet experiment, the tadpoles were divided into five groups fed respectively with five different diets. The body weight, snout-vent length and tail length were measured every seven days, and mortality was recorded. The results showed that: the survival rates of tadpole before metamorphosis and after metamorphosis were from 68.7% to 96.3% and from 5.7% to 36.0%, respectively; the optimum stocking density is 1 000 tadpoles per square meter for the stocking density had no effect on the survival rate of tadpole before metamorphosis, and the tadpoles had the relative large body weight and survival numbers in 1000 tadpoles per square meter; the diet Ⅱ(37.9% crude protein and 5.7% crude fat), Ⅳ (25.1% crude protein and 4.0% crude fat), and Ⅴ (egg yolk) were all the optimum diets for the diet had no effect on the survival rate of tadpole before metamorphosis and the tadpoles fed with three kinds of diet above had relatively large body weight, and one of these three diets based on their availability and cost should be adopted during breeding period.

[Identification and bactericidal activity of a novel Cathelicidin family member from skin of Bufu bufo gargarizans].

The skin transcriptome of Bufu bufo gargarizans was determined by conventional methods. A novel full length cDNA coding for a Cathelicidin precursor was identified by transcriptomic data assembling, annotation and blast search of corresponding data banks. According to the known processing methods of Cathelicidin family members, present reported novel Cathelicidin precursor of B. bufo gargarizans might be cleaved at 2 possible sites of the same precursor and generate both BG-CATH25 and BG-CATH29 as mature molecules. The deduced BG-CATH25 and BG-CATH29 were synthesized with purity>95% to evaluate the properties and bactericidal activities. The secondary structural characteristics of both BG-CATH25 and BG-CATH29 in different solutions were determined by Circular Dichroism (CD) Analysis. CD results indicated that random coil conformation were the main structural elements for both BG-CATH25 and BG-CATH29 in different buffer systems. Antimicrobial activities against tested bacterial strains were carried out by plating method. Both BG-CATH25 and BG-CATH29 showed strong antibacterial activities against Aeromonas hydrophila, with MIC values of 1.25, 10 mg•L⁻¹, respectively. However, both of them showed weak bactericidal activities against human pathogenic bacteria, like Escherichia coli (ATCC25922）,Staphylococcus aureus (ATCC25923）and Pseudomonas aeruginosa (ATCC 27853).

Terrestrial exposure and effects of Headline AMP(®) Fungicide on amphibians.

Recent studies have demonstrated that a pyraclostrobin-containing fungicide (Headline(®) Fungicide--Headline(®) Fungicide and Headline AMP(®) Fungicide are registered trademarks of BASF) is toxic to amphibians at environmentally relevant concentrations. However, these studies were performed in a laboratory setting of a worst-case direct exposure in clean media. Interception of spray by the crop canopy and ground cover used by animals for security cover will influence exposure. Thus, risk to amphibians is unclear in an environmentally realistic field environment. We tested exposure and toxicity of Headline AMP(®) Fungicide to amphibians in multiple agricultural habitat scenarios (e.g., within treated crop vs. grassy areas adjacent to crop) and at two rates during routine aerial application. Specifically, we placed Woodhouse's toads (Bufo woodhousii) and Blanchard's cricket frogs (Acris blanchardi) in enclosures located within treated and untreated corn (VT stage, approximate height = 3 m), and in the potential drift area (adjacent to treated corn) during aerial application of Headline AMP Fungicide at either 731 or 1052 ml/ha (70 and 100 % the maximum application rate in corn, respectively). Mean concentrations of pyraclostrobin measured at ground level were ≤19 % of nominal application rate in all areas. Overall, mean mortality of recovered individuals of both species was ≤15 %, and mortality within Headline AMP Fungicide-treated corn (where risk was anticipated to be highest) was <10 %. It is important to understand that application timing, interception by the crop canopy (which varies both within and between crop systems), and timing of amphibian presence in the crop field influences risk of exposure and effects; however, our results demonstrate that amphibians inhabiting VT stage corn during routine aerial application of Headline AMP Fungicide are at low risk for acute mortality, matching existing laboratory results from acute toxicity studies of Headline Fungicide.

Biochemical biomarkers of sublethal effects in Rhinella arenarum late gastrula exposed to the organophosphate chlorpyrifos.

We determined the biochemical and molecular effects of the organophosphate insecticide chlorpyrifos (CPF) in the late gastrula embryonic stage of the South American toad Rhinella arenarum continuously exposed from fertilization (24 h). Our objective was to evaluate these responses as potential biomarkers at low, sublethal levels of the toxicant. We first established the EC50 for embryo arrest in 21.3 mg/L, with a LOEC of 16 mg/L. At 4 mg/L CPF, some embryos were unable to complete the dorsal lip of the blastopore and the yolk plug became blur, probably because of abnormal cell migration. Acetylcholinesterase activity, the specific biomarker for organophosphates, was unaffected by any of the tested concentrations of CPF (2-14 mg/L). In turn, 2 mg/L CPF increased the reduced glutathione levels and inhibited glutathione-S-transferase activity, suggesting an oxidative stress and antioxidant response. Catalase was induced by CPF exposure at higher concentrations (8 and 14 mg/L). We also studied transcription factor c-Fos as a signaling event related to development in early embryogenesis. Analysis of nuclear c-Fos protein showed two bands, both enhanced in embryos exposed to 2 and 8 mg/L CPF. While nuclear Erk protein was practically unaffected, Mek protein levels were induced by the OP. Transcription factor c-Fos may be then linking oxidative stress with developmental alterations observed due to CPF exposure. These molecular and biochemical responses observed in R. arenarum gastrula at sublethal CPF exposures may replace non-responsive AChE as very early biomarkers in toad gastrula.

[Comparison of chemical composition between fresh and processed Bufonis Venenum by UPLC-TQ-MS].

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.