The evolution of personally disadvantageous punishment among cofoundresses of the ant Acromyrmex versicolor

Cofoundresses of the desert fungus garden ant Acromyrmex versicolorexhibit a forager specialist who subsumes all foraging risk priorto first worker eclosion (Rissing et al. 1989). In an experimentdesigned to mimic a "cheater" who refuses foraging assignment whenher lot, cofoundresses delayed/failed to replace their forager,often leading to demise of their garden (Rissing et al. 1996). Thecheater on task assignment is harmed, but so too is the punisher,as all will die without a healthy garden. In this paper we studythrough simulation the cofoundress interaction with haploid, asexualgenotypes which either replace a cheater or not (punishment), underboth foundress viscosity (likely for A. versicolor) and randomassortment. We find replacement superior to punishment only whenthere is no foraging risk and cheating is not costly to groupsurvival. Generally, punishment is evolutionarily superior,especially as forager risk increases, under both forms of dispersal.

Identification and functional characterization of Rca1, a transcription factor involved in both antifungal susceptibility and host response in Candida albicans.

The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response.

Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.

Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Epigean ant communities in Atlantic Forest remnants of São Paulo: a comparative study using the guild concept

Epigean ant communities in Atlantic Forest remnants of São Paulo: a comparative study using the guild concept. The guilds constitute a valuable ecological tool, because they allow conducting comparisons among environments under different conditions. The ants can be used as ecological indicators, mainly for the monitoring of degraded forest areas. The aim of this research was to study guild organization among the epigeous Formicidae living in Atlantic forest remnants of the State of São Paulo, Brazil. Ant collections were performed in three distinct Atlantic forest biome areas: arboreal littoral vegetation ("restinga") (Cananéia), semideciduous seasonal forest (Piracicaba) and dense ombrophylousforest (Pariquera-Açu). After identification, the ants were grouped into guilds, based on the ecological attributes of behavior and habit, according to the literature. Nine guilds were found; the semideciduous seasonal forest ecosystem presented eight of them, followed by the arboreal sandbank (7) and dense ombrophylous forest (6). The guilds found were: litter omnivorous and scavengers, granivorous species, specialist predators living in litter and soil, litter generalist predators, subterranean mealybug-dependent species, army ants, dominant or subdominants arboreal, that occasionally forage on the ground, soil or litter dominant and fungus-growers, using feces and insect body fragments. The guilds found can be used in the monitoring of the mirmecofauna in the Atlantic Forest biome, supplying insights for further ecological studies.

Mortality of Plutella xylostella (Lepidoptera, Plutellidae) by parasitoids in the Province of Santa Fe, Argentina

Mortality of Plutella xylostella (Lepidoptera, Plutellidae) by parasitoids in the Province of Santa Fe, Argentina. Plutella xylostella (Linnaeus, 1758) (Lepidoptera, Plutellidae) larvae cause severe economic damage on cabbage, Brassica oleracea L. variety capitata (Brassicaceae), in the horticultural fields in the Province of Santa Fe, Argentina. Overuse of broad spectrum insecticides affects the action of natural enemies of this insect on cabbage. The objectives of this work were to identify the parasitoids of P. xylostella and to determine their influence on larva and pupa mortality. Weekly collections of larvae and pupae were randomly conducted in cabbage crops during spring 2006 and 2007. The immature forms collected were classified according to their developmental stage: L1 and L2 (Ls = small larvae), L3 (Lm = medium larvae), L4 (Ll = large larvae), pre-pupae and pupae (P). Each individual was observed daily in the laboratory until the adult pest or parasitoid emergence. We identified parasitoids, the number of instar and the percentage of mortality of P. xylostella for each species of parasitoid. Parasitoids recorded were: Diadegma insulare (Cresson, 1875) (Hymenoptera, Ichneumonidae), Oomyzus sokolowskii (Kurdjumov, 1912) (Hymenoptera, Eulophidae), Cotesia plutellae (Kurdjumov, 1912) (Hymenoptera, Braconidae) and an unidentified species of Chalcididae (Hymenoptera). Besides parasitoids, an unidentified entomopathogenic fungus was also recorded in 2006 and 2007. In 2006, the most successful parasitoids were D. insulare and O. sokolowskii, while in 2007 only D. insulare exerted a satisfactory control and it attacked the early instars of the pest.

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The driving force behind arbuscular mycorrhizal (AM) interactions is an exchange of nutrients between fungus and plant. Glomeromycotan fungi are obligate symbionts and rely on the carbon provided by their plant hosts to complete their life cycle. In return, the fungus provides nutritional benefits to the plant, notably by delivering minerals. The majority of the nutrient exchange is thought to occur in root cortical cells containing the highly-branched fungal arbuscules. In this chapter, we describe the molecular components of the arbusculated cell and the proteins involved in the transfer of nutrients between fungus and plants. We consider, in detail, the passage of phosphorous and nitrogen from the soil to the arbusculated cell and the concomitant delivery of carbon to the fungal symbiont. In natural conditions, the exchange of nutrients does not need to be completely equitable and selective pressure may act on both partners to push the balance in their favour. In cultivated plants, the artificial environment may further distort the balance. We discuss how a better understanding of the molecular regulation of nutrient transfer benefits attempts to optimise AM associations for agriculture use.

Inflammasome Activation in Response to Eukaryotic Pathogens

Inflammasomes are multi-protein complexes that serve as platforms for caspase-1 activation and subsequent proteolytic maturation of interkeukin 1ß (IL-1ß) within innate immune cells. The Nlrp3 inflammasome is the most fully characterised. It is activated by various endogenous danger signals such as environmental irritants, signals of tissue damage and pathogens. The broad spectrum of activators is reflected at the physiological level in its implication in normal and dysregulated immune responses, including various autoinflammatory diseases and the defence agaisnt numerous pathogens. Here, we summarise the present data on the activation of the Nlrp3 inflammasome by eukaryotic pathogens. Recent genetic studies using mice deficient in inflammasome components demonstrate the involvement of the inflammasome in the outcome of infection with the fungus Candida albicans, the helminth Schistosoma mansoni, as well as the malarial parasite Plasmodium berghei. Altered immune responses were respectively linked to the ability of live fungi, schistosomal egg antigen (SEA) or malarial hemozoin to activate the inflammasome and induce secretion of mature IL-1ß. The initial findings suggest that inflammasome activation may serve as a common and potentially druggable pathway in the defence agaisnt eukaryotic pathogens

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert. The spatial distribution of the immature stages of the leaf miner Angelabella tecomae Vargas & Parra, 2005 was determined at two intra-plant levels (shoot and leaflet) on the shrub Tecoma fulva fulva (Cav.) D. Don (Bignoniaceae) in the Azapa valley, northern Chilean Atacama Desert. An aggregated spatial pattern was detected for all the immature stages along the shoot, with an age dependent relative position: eggs and first instar larvae were clumped at apex; second, third and fourth instar larvae were mostly found at intermediate positions; meanwhile the spinning larva and pupa were clumped at basis. This pattern suggests that the females select new, actively growing leaflets for egg laying. At the leaflet level, the immature stages were found more frequently at underside. Furthermore, survivorship was higher for larvae from underside mines. All these results highlight the importance of an accurate selection of egg laying site in the life history of this highly specialized leaf miner. By contrast, eventual wrong choices in the egg laying site selection may be associated with diminished larval survivorship. The importance of the continuous availability of new plant tissue in this highly human modified arid environment is discussed in relation with the observed patterns.

Growth and metabolic activity of the extramatrical mycelium of endomycorrhizal maize plants

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P.

De Novo Assembly of the Pneumocystis jirovecii Genome from a Single Bronchoalveolar Lavage Fluid Specimen from a Patient.

ABSTRACT Pneumocystis jirovecii is a fungus that causes severe pneumonia in immunocompromised patients. However, its study is hindered by the lack of an in vitro culture method. We report here the genome of P. jirovecii that was obtained from a single bronchoalveolar lavage fluid specimen from a patient. The major challenge was the in silico sorting of the reads from a mixture representing the different organisms of the lung microbiome. This genome lacks virulence factors and most amino acid biosynthesis enzymes and presents reduced GC content and size. Together with epidemiological observations, these features suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, which causes disease only in immune-deficient individuals. This genome sequence will boost research on this deadly pathogen. IMPORTANCE Pneumocystis pneumonia is a major cause of mortality in patients with impaired immune systems. The availability of the P. jirovecii genome sequence allows new analyses to be performed which open avenues to solve critical issues for this deadly human disease. The most important ones are (i) identification of nutritional supplements for development of culture in vitro, which is still lacking 100 years after discovery of the pathogen; (ii) identification of new targets for development of new drugs, given the paucity of present treatments and emerging resistance; and (iii) identification of targets for development of vaccines.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Compatibility and ectomycorrhiza formation among Pisolithus Isolates and Eucalyptus spp

Twenty-nine isolates of the ectomycorrhiza fungus Pisolithus sp. from different geographical and host origins were tested for their ability to form ectomycorrhizae on Eucalyptus grandis and E. urophylla seedlings under greenhouse conditions. The ectomycorrhiza-forming capacity of isolates varied greatly from one eucalypt species to the other. All isolates from Eucalyptus, nine from Pinus spp. and two isolates from unknown hosts formed mycorrhizae with E. grandis and E. urophylla. Root colonization rates varied from 0 to 5.2 % for all Pinus isolates and those from unknown hosts. Colonization rates for these isolates were lower than those observed for Eucalyptus isolates (0.8 to 89.4 %). Three isolates from unknown hosts formed mycorrhizae with neither Eucalyptus species. The main characteristic for distinguishing Pinus from Eucalyptus isolates was mantle color. These data corroborate previous results obtained in our laboratory indicating that the isolates tested represent at least two distinct different species within the genus Pisolithus.

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection.

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.