Ultrastructural and SINS analysis of the cell wall integrity response of Aspergillus nidulans to the absence of galactofuranose.

With lethal opportunistic fungal infections on the rise, it is imperative to explore new methods to examine virulence mechanisms. The fungal cell wall is crucial for both the virulence and viability of Aspergillus nidulans. One wall component, Galf, has been shown to contribute to important fungal processes, integrity of the cell wall and pathogenesis. Here, we explore gene deletion strains lacking the penultimate enzyme in Galf biosynthesis (ugmAΔ) and the protein that transports Galf for incorporation into the cell wall (ugtAΔ). In applying gene deletion technology to the problem of cell wall integrity, we have employed multiple micro- and nano-scale imaging tools, including confocal fluorescence microscopy, electron microscopy, X-Ray fluorescence and atomic force microscopy. Atomic force microscopy allows quantification of ultrastructural cell wall architecture while near-field infrared spectroscopy provides spatially resolved chemical signatures, both at the nanoscale. Here, for the first time, we demonstrate correlative data collection with these two emerging modalities for the multiplexed in situ study of the nanoscale architecture and chemical composition of fungal cell walls.

Overexpression of Aspergillus nidulans α-1,3-glucan synthase increases cellular adhesion and causes cell wall defects.

Alpha-1,3-glucan is important for pathogenesis by Aspergillus fumigatus, but the mechanism is unclear since the deletion has no hyphal phenotype. We dissected the roles of A. nidulans α-1,3-glucan in constitutive overexpression strains. Constitutive high-level α-1,3-glucan synthase activity increased hyphal wall thickness, but colonies grew slowly and sporulated poorly and had much higher adhesion to hydrophobic materials. Surprisingly, this overexpression strain formed a biofilm-like structure in plastic culture wells that was as adhesive as wild-type A. fumigatus. These results suggest α-1,3-glucan has important roles in fungal cellular adhesion and may contribute to fungal pathogenesis.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy.

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to the same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se0). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se0 using the Se LIII edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50-700 nm, suggesting Se0 nanoparticles. The intimate association of Se0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.

Characterization of Aspergillus nidulans α-glucan synthesis: roles for two synthases and two amylases.

Cell walls are essential for fungal survival and growth. Fungal walls are ∼ 90% carbohydrate, mostly types not found in humans, making them promising targets for anti-fungal drug development. Echinocandins, which inhibit the essential ß-glucan synthase, are already clinically available. In contrast, α-glucan, another abundant fungal cell wall component has attracted relatively little research attention because it is not essential for most fungi. Aspergillus nidulans has two α-glucan synthases (AgsA and AgsB) and two α-amylases (AmyD and AmyG), all of which affect α-glucan synthesis. Gene deletion showed that AgsB was the major synthase. In addition, AmyG promoted α-glucan synthesis whereas AmyD had a repressive effect. The lack of α-glucan had no phenotypic impact on solid medium, but reduced conidial adhesion during germination in shaken liquid. Moreover, α-glucan level correlated with resistance to Calcofluor White. Intriguingly, overexpression of agsA could compensate for the loss of agsB at the α-glucan level, but not for phenotypic defects. Thus, products of AgsA and AgsB have different roles in the cell wall, consistent with agsA being mainly expressed at conidiation. These results suggest that α-glucan contributes to drug sensitivity and conidia adhesion in A. nidulans, and is differentially regulated by two synthases and two amylases.

Using Aspergillus nidulans to identify antifungal drug resistance mutations.

Systemic fungal infections contribute to at least 10% of deaths in hospital settings. Most antifungal drugs target ergosterol (polyenes) or its biosynthetic pathway (azoles and allylamines), or beta-glucan synthesis (echinocandins). Antifungal drugs that target proteins are prone to the emergence of resistant strains. Identification of genes whose mutations lead to targeted resistance can provide new information on those pathways. We used Aspergillus nidulans as a model system to exploit its tractable sexual cycle and calcofluor white as a model antifungal agent to cross-reference our results with other studies. Within 2 weeks from inoculation on sublethal doses of calcofluor white, we isolated 24 A. nidulans adaptive strains from sectoring colonies. Meiotic analysis showed that these strains had single-gene mutations. In each case, the resistance was specific to calcofluor white, since there was no cross-resistance to caspofungin (echinocandin). Mutation sites were identified in two mutants by next-generation sequencing. These were confirmed by reengineering the mutation in a wild-type strain using a gene replacement strategy. One of these mutated genes was related to cell wall synthesis, and the other one was related to drug metabolism. Our strategy has wide application for many fungal species, for antifungal compounds used in agriculture as well as health care, and potentially during protracted drug therapy once drug resistance arises. We suggest that our strategy will be useful for keeping ahead in the drug resistance arms race.

X-ray microfluorescence (µXRF) imaging of Aspergillus nidulans cell wall mutants reveals biochemical changes due to gene deletions.

We used synchrotron X-ray fluorescence to create the first semiquantitative, submicron resolution, element distribution maps of P, S, K, and Ca, in situ, in fungal samples. Data collection was performed at the European Synchrotron Radiation Facility beam line ID21, Grenoble, France. We studied developing hyphae, septa, and conidiophores in Aspergillus nidulans, comparing wild type and two cell wall biosynthesis gene deletion strains. The latter encode sequential enzymes for biosynthesis of galactofuranose, a minor wall carbohydrate. Each gene deletion caused hyphal morphogenesis defects, and reduced both colony growth and sporulation 500-fold. Elemental imaging has helped elucidate biochemical changes in the phenotype induced by the gene deletions that were not apparent from morphological examination. Here, we examined S as a proxy for protein content, P for nucleic acid content, as well as Ca and K, which also have important metabolic roles. Element distributions in wild-type fungi reflect biological aspects already known or expected from other types of analysis; however, the application of X-ray fluorescence (XRF) imaging reveals aspects of gene deletion phenotypes that were not previously available. We have demonstrated that deleting a dispensable gene involved in galactose metabolism (ugeA) and one involved in biosynthesis of a minor cell wall component (ugmA) led to changes in hyphal elemental distribution that may have resulted from compromised wall composition.

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity.

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.

Proof-of-principle for SERS imaging of Aspergillus nidulans hyphae using in vivo synthesis of gold nanoparticles.

High spatial resolution methods to assess the physiology of growing cells should permit analysis of fungal biochemical composition. Whole colony methods cannot capture the details of physiology and organism-environment interaction, in part because the structure, function and composition of fungal hyphae vary within individual cells depending on their distance from the growing apex. Surface Enhanced Raman Scattering (SERS) can provide chemical information on materials that are in close contact with appropriate metal substrates, such as nanopatterned gold surfaces and gold nanoparticles (AuNPs). Since nanoparticles can be generated by living cells, we have created conditions for AuNP formation within and on the surface of Aspergillus nidulans hyphae in order to explore their potential for SERS analysis. AuNP distribution and composition have been assessed by UV-Vis spectroscopy, fluorescence light microscopy, transmission electron microscopy, and scanning transmission X-ray microscopy. AuNPs were often associated with hyphal walls, both in the peripheral cytoplasm and on the outer wall surface. Interpretation of SERS spectra is challenging, and will require validation for the diversity of organic molecules present. Here, we show proof-of-principle that it is possible to generate SERS spectra from nanoparticles grown in situ by living hyphae.

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity.

Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Galf from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Galf residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Galf transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. AfglfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Galf, which was restored in the AfglfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.

Preliminary X-ray crystallographic studies of UDP-glucose-4-epimerase from Aspergillus nidulans.

UDP-glucose-4-epimerase (GALE) from Aspergillus nidulans was overexpressed in Escherichia coli, purified via His-tag affinity chromatography and cocrystallized with UDP-galactose using the microbatch method. The crystals diffracted to 2.4 Šresolution using synchrotron radiation on the Canadian Light Source 08ID-1 beamline. Examination of the data with d*TREK revealed nonmerohedral twinning, from which a single lattice was ultimately extracted for processing. The final space group was found to be C2, with unit-cell parameters a = 66.13, b = 119.15, c = 161.42 Å, ß = 98.48°. An initial structure solution has been obtained via molecular replacement employing human GALE (PDB entry 1hzj) as a template model.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeA Delta strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmA Delta strain. As for the ugmA Delta strain, ugeA Delta colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeA Delta strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeA Delta, and ugmA Delta hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmA Delta strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.

Characterization of mannitol in Curvularia protuberata hyphae by FTIR and Raman spectromicroscopy.

FTIR and Raman spectromicroscopy were used to characterize the composition of Curvularia protuberata hyphae, and to compare a strain isolated from plants inhabiting geothermal soils with a non-geothermal isolate. Thermal IR source images of hyphae have been acquired with a 64 × 64 element focal plane array detector; single point IR spectra have been obtained with synchrotron source light. In some C. protuberata hyphae, we have discovered the spectral signature of crystalline mannitol, a fungal polyol with complex protective roles. With FTIR-FPA imaging, we have determined that the protein content in cells remains fairly constant throughout the length of a hypha, whereas the mannitol is found at discrete, irregular locations. This is the first direct observation of mannitol in intact fungal hyphae. Since the concentration of mannitol in cells varies with respect to position and is not present in all hyphae, this discovery may be related to habitat adaptation, fungal structure and growth stages.

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogenesis, and conidiation.

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.

Aspergillus nidulans hypB encodes a Sec7-domain protein important for hyphal morphogenesis.

Aspergillus nidulans strains containing the hypB5 temperature sensitive allele have a restrictive phenotype of wide, highly-branched hyphae. The hypB locus was cloned by phenotype complementation using a genomic plasmid library. hypB5 is predicted gene AN6709 in the A. nidulans genome database, which encodes a putative Sec7 domain protein, likely to act early in COPI-mediated vesicle formation for retrograde Golgi to ER transport. The A. nidulans hypB5 allele has a single mutation, cytosine to guanine predicted to cause a nonconservative amino acid change, alanine to proline, in a conserved helix adjacent to the Sec7p nucleotide binding site. This would likely reduce the stability of a highly conserved loop important for nucleotide binding, and is consistent with temperature sensitivity of hypB5 strains. Deletion of AN6709 showed that hypB(Sec7) was not essential. AN6709Delta hyphae resembled the hypB5 restrictive phenotype. As has been shown previously for hypA1 mutants, shifting established hypB5 mutant hyphae from a growth temperature of 28-42 degrees C caused septation in and death of tip cells and growth activation of basal cells.

A sensitive method for examining whole-cell biochemical composition in single cells of filamentous fungi using synchrotron FTIR spectromicroscopy.

Cell function is related to cell composition. The asexual state of filamentous fungi (molds and mildews) has two main life cycle stages: vegetative hyphae for substrate colonization and nutrient acquisition, and asexual spores for survival and dispersal. Hyphal composition changes over a few tens of microns during growth and maturation; spores are different from hyphae. Most biochemical analyses are restricted to studying a few components at high spatial resolution (e.g. histochemistry) or many compounds at low spatial resolution (e.g. GC-MS). Synchrotron FTIR spectromicroscopy can be used to study fungal cell biology by fingerprinting varieties of carbohydrates, proteins, and lipids at about 6 microm spatial resolution. FTIR can distinguish fungal species and changes during hyphal growth, and reveals that even fungi grown under optimal vs mildly stressed conditions exhibit dramatic biochemical changes without obvious morphological effects. Here we compare hypha and spore composition of two fungi, Neurospora and Rhizopus. There are clear biochemical changes when Neurospora hyphae commit to spore development, during spore maturation and following germination, many of which are consistent with results from molecular genetics, but have not been shown before at high spatial resolution. Rhizopus spores develop within a fluid-containing sporangium that becomes dry at maturity. Rhizopus spores had similar protein content and significantly more carbohydrate than the sporangial fluid, both of which are novel findings.

High spatial resolution surface imaging and analysis of fungal cells using SEM and AFM.

We review the use of scanning electron microscopy (SEM), atomic force microscopy (AFM) and force spectroscopy (FS) for probing the ultrastructure, chemistry, physical characteristics and motion of fungal cells. When first developed, SEM was used to image fixed/dehydrated/gold coated specimens, but here we describe more recent SEM developments as they apply to fungal cells. CryoSEM offers high resolution for frozen fungal samples, whereas environmental SEM allows the analysis of robust samples (e.g. spores) under ambient conditions. Dual beam SEM, the most recently developed, adds manipulation capabilities along with element detection. AFM has similar lateral and better depth resolution compared to SEM, and can image live cells including growing fungal hyphae. FS can analyze cell wall chemistry, elasticity and dynamic cell characteristics. The integration of AFM with optical microscopy will allow examination of individual molecules or cellular structures in the context of fungal cell architecture. SEM and AFM are complementary techniques that are clarifying our understanding of fungal biology.

Epidermal 'alarm substance' cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation.

Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.