First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Comparative insights of the neuroanatomical distribution of the gonadotropin-inhibitory hormone (GnIH) in fish and amphibians.

This paper intends to apprise the reader regarding the existing knowledge on the neuroanatomical distribution of GnIH-like peptides in in fish and amphibians in both the adult stage and during ontogenesis. The neuroanatomical distribution of GnIH-like neuropeptides appears quite different in the studied species, irrespective of the evolutionary closeness. The topology of the olfactory bulbs can affect the distribution of neurons producing the GnIH-like peptides, with a tendency to show a more extended distribution into the brains with pedunculate olfactory bulbs. Therefore, the variability of the GnIH-like system could also reflect specific adaptations rather than evolutionary patterns. The onset of GnIH expression was detected very early during development suggesting its precocious roles, and the neuroanatomical distribution of GnIH-like elements showed a generally increasing trend. This review highlights some critical technical aspects and the need to increase the number of species to be studied to obtain a complete neuroanatomical picture of the GnIH-like system.

Complex Life Histories Alter Patterns of Mercury Exposure and Accumulation in a Pond-Breeding Amphibian.

Quantifying how contaminants change across life cycles of species that undergo metamorphosis is critical to assessing organismal risk, particularly for consumers. Pond-breeding amphibians can dominate aquatic animal biomass as larvae and are terrestrial prey as juveniles and adults. Thus, amphibians can be vectors of mercury exposure in both aquatic and terrestrial food webs. However, it is still unclear how mercury concentrations are affected by exogenous (e.g., habitat or diet) vs endogenous factors (e.g., catabolism during hibernation) as amphibians undergo large diet shifts and periods of fasting during ontogeny. We measured total mercury (THg), methylmercury (MeHg), and isotopic compositions (δ 13C, δ15N) in boreal chorus frogs (Pseudacris maculata) across five life stages in two Colorado (USA) metapopulations. We found large differences in concentrations and percent MeHg (of THg) among life stages. Frog MeHg concentrations peaked during metamorphosis and hibernation coinciding with the most energetically demanding life cycle stages. Indeed, life history transitions involving periods of fasting coupled with high metabolic demands led to large increases in mercury concentrations. The endogenous processes of metamorphosis and hibernation resulted in MeHg bioamplification, thus decoupling it from the light isotopic proxies of diet and trophic position. These step changes are not often considered in conventional expectations of how MeHg concentrations within organisms are assessed.

Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian.

In the Paraná River lower basin, an important agro-productive area of Argentina, crop fields and cattle breeding activities are common and may affect water quality. So, the aim of this study was to analyze the impacts of cattle breeding and agricultural activities on a stream from Buenos Aires, through physicochemical parameters (metals, pesticides, and emerging contaminants) and ecotoxicological parameters with Rhinella arenarum larvae, a native amphibian species. Three sites were selected on an ordinary plain stream that goes through agricultural fields and a cattle breeding establishment (upstream -S1-, near -S2- and downstream -S3- the establishment). Physicochemical parameters were measured in situ (in water) and in laboratory (in water and sediment samples: metals, pesticides, ivermectin and oxytetracycline). A semi-static chronic toxicity bioassay (504 h) was performed with water samples, and neurotoxicity, oxidative stress and genotoxicity biomarkers were measured after acute exposure (96 h). According to the index, a degradation in the water quality was observed in all sites. Ivermectin (8.03 mg/kg) and oxytetracycline (1.9 mg/kg) were detected in sediment samples from S2. Pesticides were detected in all sites, mainly in water samples: S1 presented the highest variability (7 residues) and in S3 AMPA, glyphosate and acetochlor concentrations were higher (10.3, 22.4 and 23.8 µg/L). Also, all sites significantly produced lethality at chronic exposure. Lethality at 504h was 40% for S1, 56.66% for S2 and 93.33% for S3. At acute exposure, the oxidative stress biomarkers were altered on R. arenarum larvae exposed to all sites and the neurotoxicity biomarkers were altered on larvae exposed to S1 and S3. Water quality was severely degraded by the surrounding agricultural and cattle breeding activities, which may represent a threat to the ecosystems.

MeDAS: a Metazoan Developmental Alternative Splicing database.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively.

What snakes and caecilians have in common? Molecular interaction units and the independent origins of similar morphotypes in Tetrapoda.

Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.

Keratinization and Cornification are not equivalent processes but keratinization in fish and amphibians evolved into cornification in terrestrial vertebrates.

The present account offers a generalized view of the evolution of process of terminal differentiation in keratinocytes of the epidermis in anamniotes, indicated as keratinization, into a further differentiating process of cornification in the skin and appendages of terrestrial vertebrates. Keratinization indicates the prevalent accumulation of intermediate filaments of keratins (IFKs) and is present in most fish and amphibian epidermis and inner epithelia of all vertebrates. During land adaptation, terrestrial vertebrates evolved a process of cornification and keratinocytes became dead corneocytes by the addition of numerous others proteins to the IFKs framework, represented by keratin-associated proteins (KAPs) and corneous proteins (CPs). Most of genes coding for these types of proteins are localized in chromosomal loci different and un-related from those of IFKs, and CPs originated from a gene cluster indicated as epidermal differentiation complex. During the evolution of reptiles and birds, the epidermis and corneous derivatives such as scales, claws, beaks and feathers mainly accumulate a type of CPs that overcome IFKs and containing a 34 amino acid beta-sheet core indicated as corneous beta proteins, formerly known as beta-keratins. Mammals did not evolve a beta-sheet core in their CPs and KAPs but instead produced numerous cysteine-rich IFKs in their epidermis and specialized KAPs in hairs, claws, nails, hooves and horns.

Occurrence and Distribution of Persistent Organic Pollutants (POPs) in Amphibian Species: Implications from Biomagnification Factors Based on Quantitative Fatty Acid Signature Analysis.

Contaminants pose a great threat to amphibian populations, but the bioaccumulation and distribution of contaminants in amphibians are still unclear. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) had median concentrations of 468-3560 ng/g lipid weight (lw) and 206-2720 ng/g lw in the muscle of amphibians, respectively. BDE 209 was the predominant PBDE congener, while CBs 118, 138, 153, and 180 were the main PCB congeners. The diet compositions of amphibians were estimated by quantitative fatty acid signature analysis (QFASA). Dragonfly contributed the most to the diet of amphibians. Biomagnification factors (BMFs) based on quantitative amphibian/insect relationships showed more credible results than BMFs based on amphibian/each insect or amphibian/combined prey relationships. BMFs derived from QFASA declined with log KOW from 5 to 6.5 and then showed a parabolic relationship with log KOW greater than 6.5. BMFs of PCBs were significantly influenced by the elimination capacity of PCBs in amphibians. Less-hydrophobic PCBs preferentially accumulated in the skin than in muscle, which was probably due to the dermal exposure of less-hydrophobic PCBs for amphibians. The biomagnification and distribution of contaminants may be affected by multiple exposure pathways and the toxicokinetics of contaminants in various life stages of amphibians.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Nerve-associated Schwann cell precursors contribute extracutaneous melanocytes to the heart, inner ear, supraorbital locations and brain meninges.

Melanocytes are pigmented cells residing mostly in the skin and hair follicles of vertebrates, where they contribute to colouration and protection against UV-B radiation. However, the spectrum of their functions reaches far beyond that. For instance, these pigment-producing cells are found inside the inner ear, where they contribute to the hearing function, and in the heart, where they are involved in the electrical conductivity and support the stiffness of cardiac valves. The embryonic origin of such extracutaneous melanocytes is not clear. We took advantage of lineage-tracing experiments combined with 3D visualizations and gene knockout strategies to address this long-standing question. We revealed that Schwann cell precursors are recruited from the local innervation during embryonic development and give rise to extracutaneous melanocytes in the heart, brain meninges, inner ear, and other locations. In embryos with a knockout of the EdnrB receptor, a condition imitating Waardenburg syndrome, we observed only nerve-associated melanoblasts, which failed to detach from the nerves and to enter the inner ear. Finally, we looked into the evolutionary aspects of extracutaneous melanocytes and found that pigment cells are associated mainly with nerves and blood vessels in amphibians and fish. This new knowledge of the nerve-dependent origin of extracutaneous pigment cells might be directly relevant to the formation of extracutaneous melanoma in humans.

Nicosulfuron, a sulfonylurea herbicide, alters embryonic development and oxidative status of hatchlings at environmental concentrations in an amphibian species.

The widespread use of agrochemicals for controlling pests and diseases of crops is recognized as a main threat to biodiversity. Sulfonylurea herbicides are being increasingly used and display low levels of degradation in water which suggest that they might affect non-target organisms. In a common garden experiment, eggs of a widespread amphibian (Bufo spinosus) were exposed to sublethal environmentally relevant concentrations of a widely used sulfonylurea herbicide, nicosulfuron, during the whole embryonic development. We assessed development-related traits (i.e., development duration, hatching success, hatchling size and occurrence of malformation) as well as antioxidant markers in response to contamination (i.e., SOD, GPx, catalase, thiols and relevant ratios thereof). We found that sublethal concentrations of nicosulfuron increased embryonic development duration, increased hatchling size and tended to increase malformations. Embryos exposed to nicosulfuron displayed decreased thiols and increased catalase activity suggesting alteration of oxidative status. We did not find any effect of nicosulfuron on SOD and GPx levels. Interestingly, higher catalase activity was linked to higher proportion of malformed individuals, suggesting that exposure to nicosulfuron induced teratogenic effects. Our results suggest that alteration of antioxidant levels might be one physiological mechanism through which nicosulfuron might cause detrimental effects on amphibian embryos. Sublethal effects of pesticides at environmentally relevant concentrations have been overlooked and require further investigations, especially in non-target taxa occurring in agricultural landscapes.

Amyloid Properties of the FXR1 Protein Are Conserved in Evolution of Vertebrates.

Functional amyloids are fibrillary proteins with a cross-ß structure that play a structural or regulatory role in pro- and eukaryotes. Previously, we have demonstrated that the RNA-binding FXR1 protein functions in an amyloid form in the rat brain. This RNA-binding protein plays an important role in the regulation of long-term memory, emotions, and cancer. Here, we evaluate the amyloid properties of FXR1 in organisms representing various classes of vertebrates. We show the colocalization of FXR1 with amyloid-specific dyes in the neurons of amphibians, reptiles, and birds. Moreover, FXR1, as with other amyloids, forms detergent-resistant insoluble aggregates in all studied animals. The FXR1 protein isolated by immunoprecipitation from the brains of different vertebrate species forms fibrils, which show yellow-green birefringence after staining with Congo red. Our data indicate that in the evolution of vertebrates, FXR1 acquired amyloid properties at least 365 million years ago. Based on the obtained data, we discuss the possible role of FXR1 amyloid fibrils in the regulation of vital processes in the brain of vertebrates.

Broad-Spectrum Antiviral Activity of the Amphibian Antimicrobial Peptide Temporin L and Its Analogs.

The COVID-19 pandemic has evidenced the urgent need for the discovery of broad-spectrum antiviral therapies that could be deployed in the case of future emergence of novel viral threats, as well as to back up current therapeutic options in the case of drug resistance development. Most current antivirals are directed to inhibit specific viruses since these therapeutic molecules are designed to act on a specific viral target with the objective of interfering with a precise step in the replication cycle. Therefore, antimicrobial peptides (AMPs) have been identified as promising antiviral agents that could help to overcome this limitation and provide compounds able to act on more than a single viral family. We evaluated the antiviral activity of an amphibian peptide known for its strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, namely Temporin L (TL). Previous studies have revealed that TL is endowed with widespread antimicrobial activity and possesses marked haemolytic activity. Therefore, we analyzed TL and a previously identified TL derivative (Pro3, DLeu9 TL, where glutamine at position 3 is replaced with proline, and the D-Leucine enantiomer is present at position 9) as well as its analogs, for their activity against a wide panel of viruses comprising enveloped, naked, DNA and RNA viruses. We report significant inhibition activity against herpesviruses, paramyxoviruses, influenza virus and coronaviruses, including SARS-CoV-2. Moreover, we further modified our best candidate by lipidation and demonstrated a highly reduced cytotoxicity with improved antiviral effect. Our results show a potent and selective antiviral activity of TL peptides, indicating that the novel lipidated temporin-based antiviral agents could prove to be useful additions to current drugs in combatting rising drug resistance and epidemic/pandemic emergencies.

Codon usage pattern and evolutionary forces of mitochondrial ND genes among orders of class Amphibia.

In this study, we used a bioinformatics approach to analyze the nucleotide composition and pattern of synonymous codon usage in mitochondrial ND genes in three amphibian groups, that is, orders Anura, Caudata, and Gymnophiona to identify the commonality and the differences of codon usage as no research work was reported yet. The high value of the effective number of codons revealed that the codon usage bias (CUB) was low in mitochondrial ND genes among the orders. Nucleotide composition analysis suggested that for each gene, the compositional features differed among Anura, Caudata, and Gymnophiona and the GC content was lower than AT content. Furthermore, a highly significant difference (p < .05) for GC content was found in each gene among the orders. The heat map showed contrasting patterns of codon usage among different ND genes. The regression of GC12 on GC3 suggested a narrow range of GC3 distribution and some points were located in the diagonal, indicating both mutation pressure and natural selection might influence the CUB. Moreover, the slope of the regression line was less than 0.5 in all ND genes among orders, indicating natural selection might have played the dominant role whereas mutation pressure had played a minor role in shaping CUB of ND genes across orders.

POPULATION PHARMACOKINETICS OF CEFTAZIDIME AFTER A SINGLE SUBCUTANEOUS INJECTION AND NORMAL ORAL AND CLOACAL BACTERIAL FLORA SURVEY IN EASTERN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS ALLEGANIENSIS).

Population pharmacokinetics utilizing sparse sampling were used to determine pharmacokinetics of ceftazidime in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) due to their slow growth rate and the limited number of appropriately sized individuals in the zoo-housed population. Twenty-five eastern hellbenders received a single subcutaneous injection of ceftazidime at 20 mg/kg. Each animal had blood samples collected up to four times between 0 and 192 hr postinjection. Plasma samples were analyzed by high-pressure liquid chromatography. A nonlinear mixed-effects model was fitted to the data to determine typical values for population parameters, an ideal method due to the sampling limitation of each hellbender. Results indicate an elimination half-life of 36.63 hr and volume of distribution of 0.31 L/kg. Antibiotic concentrations were above a minimum inhibitory concentration (MIC) value of 8 µg/ml for 120 hr. Prior to antibiotic administration, six hellbenders had oral and six other individuals had cloacal swabs taken for aerobic culture. Fifty-five bacterial isolates were obtained (24 cloacal, 31 oral) with 10/12 (83%) individuals growing three or more different isolates and 11/12 (92%) growing Shewanella putrefaciens. Twelve isolates had susceptibility testing performed and all were susceptible to ceftazidime. These results indicate that ceftazidime is an appropriate choice of antibiotic in hellbenders and when given at a dosage of 20 mg/kg subcutaneously, maintains concentrations above the MIC of susceptible bacteria for up to 5 days.

Pyridine alkaloids with activity in the central nervous system.

This review discusses all pyridine alkaloids with CNS activity, their therapeutic potential, and the interesting array of sources whence they originate.

Relationships between glucocorticoids and infection with Batrachochytrium dendrobatidis in three amphibian species.

It is often hypothesized that organisms exposed to environmental change may experience physiological stress, which could reduce individual quality and make them more susceptible to disease. Amphibians are amongst the most threatened taxa, particularly in the context of disease, but relatively few studies explore links between stress and disease in amphibian species. Here, we use the fungal pathogen Batrachochytrium dendrobatidis (Bd) and amphibians as an example to explore relationships between disease and glucocorticoids (GCs), metabolic hormones that comprise one important component of the stress response. While previous work is limited, it has largely identified positive relationships between GCs and Bd-infection. However, the causality remains unclear and few studies have integrated both baseline (GC release that is related to standard, physiological functioning) and stress-induced (GC release in response to an acute stressor) measures of GCs. Here, we examine salivary corticosterone before and after exposure to a stressor, in both field and captive settings. We present results for Bd-infected and uninfected individuals of three amphibian species with differential susceptibilities to this pathogen (Rana catesbeiana, R. clamitans, and R. sylvatica). We hypothesized that prior to stress, baseline GCs would be higher in Bd-infected animals, particularly in more Bd-susceptible species. We also expected that after exposure to a stressor, stress-induced GCs would be lower in Bd-infected animals. These species exhibited significant interspecific differences in baseline and stress induced corticosterone, though other variables like sex, body size, and day of year were usually not predictive of corticosterone. In contrast to most previous work, we found no relationships between Bd and corticosterone for two species (R. catesbeiana and R. clamitans), and in the least Bd-tolerant species (R. sylvatica) animals exhibited context-dependent differences in relationships between Bd infection and corticosterone: Bd-positive R. sylvatica had significantly lower baseline and stress-induced corticosterone, with this pattern being stronger in the field than in captivity. These results were surprising, as past work in other species has more often found elevated GCs in Bd-positive animals, a pattern that aligns with well-documented relationships between chronically high GCs, reduced individual quality, and immunosuppression. This work highlights the potential relevance of GCs to disease susceptibility in the context of amphibian declines, while underscoring the importance of characterizing these relationships in diverse contexts.

Antiviral peptides as promising therapeutic drugs.

While scientific advances have led to large-scale production and widespread distribution of vaccines and antiviral drugs, viruses still remain a major cause of human diseases today. The ever-increasing reports of viral resistance and the emergence and re-emergence of viral epidemics pressure the health and scientific community to constantly find novel molecules with antiviral potential. This search involves numerous different approaches, and the use of antimicrobial peptides has presented itself as an interesting alternative. Even though the number of antimicrobial peptides with antiviral activity is still low, they already show immense potential to become pharmaceutically available antiviral drugs. Such peptides can originate from natural sources, such as those isolated from mammals and from animal venoms, or from artificial sources, when bioinformatics tools are used. This review aims to shed some light on antimicrobial peptides with antiviral activities against human viruses and update the data about the already well-known peptides that are still undergoing studies, emphasizing the most promising ones that may become medicines for clinical use.

Introducing the Amphibious Mudskipper Goby as a Unique Model to Evaluate Neuro/Endocrine Regulation of Behaviors Mediated by Buccal Sensation and Corticosteroids.

Some fish have acquired the ability to breathe air, but these fish can no longer flush their gills effectively when out of water. Hence, they have developed characteristic means for defense against external stressors, including thirst (osmolarity/ions) and toxicity. Amphibious fish, extant air-breathing fish emerged from water, may serve as models to examine physiological responses to these stressors. Some of these fish, including mudskipper gobies such as Periophthalmodon schlosseri, Boleophthalmus boddarti and our Periophthalmus modestus, display distinct adaptational behaviors to these factors compared with fully aquatic fish. In this review, we introduce the mudskipper goby as a unique model to study the behaviors and the neuro/endocrine mechanisms of behavioral responses to the stressors. Our studies have shown that a local sensation of thirst in the buccal cavity-this being induced by dipsogenic hormones-motivates these fish to move to water through a forebrain response. The corticosteroid system, which is responsive to various stressors, also stimulates migration, possibly via the receptors in the brain. We suggest that such fish are an important model to deepen insights into the stress-related neuro/endocrine-behavioral effects.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.