Towards a molecular understanding of symbiont function: Identification of a fungal gene for the degradation of xylan in the fungus gardens of leaf-cutting ants

<p>Abstract</p> <p>Background</p> <p>Leaf-cutting ants live in symbiosis with a fungus that they rear for food by providing it with live plant material. Until recently the fungus' main inferred function was to make otherwise inaccessible cell wall degradation products available to the ants, but new studies have shed doubt on this idea. To provide evidence for the cell wall degrading capacity of the attine ant symbiont, we designed PCR primers from conserved regions of known xylanase genes, to be used in PCR with genomic DNA from the symbiont as template. We also measured xylanase, cellulase and proteinase activities in the fungus gardens in order to investigate the dynamics of degradation activities.</p> <p>Results</p> <p>We cloned a xylanase gene from the mutualistic fungus of <it>Acromyrmex echinatior</it>, determined its protein sequence, and inserted it in a yeast expression vector to confirm its substrate specificity. Our results show that the fungus has a functional xylanase gene. We also show by lab experiments <it>in vivo </it>that the activity of fungal xylanase and cellulase is not evenly distributed, but concentrated in the lower layer of fungus gardens, with only modest activity in the middle layer where gongylidia are produced and intermediate activity in the newly established top layer. This vertical distribution appears to be negatively correlated with the concentration of glucose, which indicates a directly regulating role of glucose, as has been found in other fungi and has been previously suggested for the ant fungal symbiont.</p> <p>Conclusion</p> <p>The mutualistic fungus of <it>Acromyrmex echinatior </it>has a functional xylanase gene and is thus presumably able to at least partially degrade the cell walls of leaves. This finding supports a saprotrophic origin of the fungal symbiont. The observed distribution of enzyme activity leads us to propose that leaf-substrate degradation in fungus gardens is a multi-step process comparable to normal biodegradation of organic matter in soil ecosystems, but with the crucial difference that a single fungal symbiont realizes most of the steps that are normally provided by a series of microorganisms that colonize fallen leaves in a distinct succession.</p

Comparative RNAseq Analysis of the Insect-Pathogenic Fungus <i>Metarhizium anisopliae</i> Reveals Specific Transcriptome Signatures of Filamentous and Yeast-Like Development

The fungus Metarhizium anisopliae is a facultative insect pathogen used as biological control agent of several agricultural pests worldwide. It is a dimorphic fungus that is able to display two growth morphologies, a filamentous phase with formation of hyphae and a yeast-like phase with formation of single-celled blastospores. Blastospores play an important role for M. anisopliae pathogenicity during disease development. They are formed solely in the hemolymph of infected insects as a fungal strategy to quickly multiply and colonize the insect’s body. Here, we use comparative genome-wide transcriptome analyses to determine changes in gene expression between the filamentous and blastospore growth phases in vitro to characterize physiological changes and metabolic signatures associated with M. anisopliae dimorphism. Our results show a clear molecular distinction between the blastospore and mycelial phases. In total 6.4% (n = 696) out of 10,981 predicted genes in M. anisopliae were differentially expressed between the two phases with a fold-change > 4. The main physiological processes associated with up-regulated gene content in the single-celled yeast-like blastospores during liquid fermentation were oxidative stress, amino acid metabolism (catabolism and anabolism), respiration processes, transmembrane transport and production of secondary metabolites. In contrast, the up-regulated gene content in hyphae were associated with increased growth, metabolism and cell wall re-organization, which underlines the specific functions and altered growth morphology of M. anisopliae blastospores and hyphae, respectively. Our study revealed significant transcriptomic differences between the metabolism of blastospores and hyphae. These findings illustrate important aspects of fungal morphogenesis in M. anisopliae and highlight the main metabolic activities of each propagule under in vitro growth conditions

Phylogenetic analyses of diverse <i>Podaxis</i> specimens from Southern Africa reveal hidden diversity and new insights into associations with termites

<p>Although frequently found on mounds of the grass-cutting termite genus . Trinervitermes, virtually nothing is known about the natural history of the fungal genus . Podaxis (Agaricaceae) nor why it associates with termite mounds. More than 40 species of this secotioid genus have been described since Linnaeus characterised the first species in 1771. However, taxonomic confusion arose when most of these species were reduced to synonymy with . Podaxis pistillaris in 1933. Although a few more species have since been described, the vast majority of specimens worldwide are still treated as . P. pistillaris. Using 45 fresh and herbarium specimens from Southern Africa, four from North America and one each from Ethiopia, and Kenya, we constructed the first comprehensive phylogeny of the genus. Four of the genotyped specimens were more than 100 y old. With the exception of the type specimen of . Podaxis rugospora, all herbarium specimens were labelled as . P. pistillaris or . Podaxis sp. However, our data shows that the genus contains at least five well-supported clades with significant inter-clade differences in spore length, width and wall thickness, and fruiting body length, supporting that clades likely represent distinct . Podaxis species. Certain clades consistently associate with termites while others appear entirely free-living.</p

Molecular adaptations to advanced fungus farming in leaf-cutting ant symbiosis

This paper addresses several aspects of legal regulation concerning cosmetics, homeopathy products and medical devices. The Portuguese legal framework, based mainly upon European directives, is analyzed concerning the administrative legal environment of the production, distribution and marketing of these products. It is stressed that legislation aims to achieve a balance between the values of free trade and enterprise, on one side, and the protection of public health protection, on the other side.1. Cosméticos – Regime jurídico dos cosméticos (Decreto-Lei n.º 296/98, de 25 de Setembro; Decreto-Lei n.º 100/2001, de 28 de Março; Decreto-Lei n.º 206/99, de 9 de Junho). 1.1. Noção funcional de produtos cosméticos e de higiene corporal, ilustrada mediante uma lista (indicativa) de exemplos por categorias de produtos cosméticos e de higiene corporal. 1.2. Desnecessidade de obtenção de autorização administrativa prévia, mas dever de notificação ao INFARMED. 1.3. Requisitos de qualidade e regras de composição e de experimentação (Decreto-Lei n.º 100/2001, de 28 de Março, alterado pelo Decreto-Lei n.º 151/2003, de 11 de Julho). 1.4. Obrigação de assistência por um técnico responsável. 1.5. Rotulagem. 1.6. Requisitos da actividade industrial. 1.7. A protecção da confidencialidade (Decreto-Lei n.º 206/99, de 9 de Junho). 1.8. Sanções. 1.8.1. Poderes de controlo e fiscalização do INFARMED. 1.8.2. Suspensão da comercialização dos produtos por razões de saúde pública. 1.8. 3. As contra-ordenações 2. Produtos Homeopáticos. 2.1. Linhas gerais do Regime jurídico dos produtos homeopáticos (Decreto-Lei n.º 94/95, de 9 de Maio). 2.1.1. Garantia da qualidade e da segurança de utilização dos produtos homeopáticos como salvaguarda da saúde pública.2.1.2. Garantia aos seus utilizadores do fornecimento de informações claras sobre o seu carácter homeopático e a sua inocuidade). 2.2. Noção e modalidades de produtos homeopáticos. 2.2.1. Medicamentos homeopáticos. 2.2.2. Produtos farmacêuticos homeopáticos. 2.3. Delimitação do âmbito de aplicação da lei dos produtos homeopáticos aos produtos farmacêuticos homeopáticos e aplicação do regime jurídico dos medicamentos para uso humano (Decreto-Lei n.º 72/91, 8.2) aos medicamentos homeopáticos. 2.3.1. Comercialização de medicamentos homeopáticos entre fabricantes, grossistas, laboratórios e farmácias. 2.3.2. Venda de medicamentos homeopáticos ao público. 2.4. Regime de registo simplificado da introdução no mercado dos produtos farmacêuticos homeopáticos. 2.4.1. O pedido de registo. 2.4.2. Necessidade de autorização para o fabrico de produtos farmacêuticos homeopáticos. 2.4.3. Exigência de direcção técnica. 2.4.4. Requisitos relativos à rotulagem e ao folheto informativo. 2.5. Fiscalização e contra-ordenações. 3. Dispositivos Médicos – Regime jurídico dos dispositivos médicos (Decreto-Lei n.º 273/95, de 23 de Outubro, alterado pelo Decreto-Lei n.º 30/2003, de 14 de Fevereiro). 3.1. Noção e modalidades de dispositivos médicos. 3.2. Delimitação positiva e negativa do âmbito de aplicação do regime geral dos dispositivos médicos; regimes especiais, como o dos dispositivos médicos para diagnóstico in vitro (Decreto-Lei n.º 189/2000, de 12 de Agosto - que transpõe a Directiva 98/79/CE do Parlamento Europeu e do Conselho, de 27 de Outubro). 3.3. Requisitos de colocação no mercado. 3.3.1 As normas técnicas e os procedimentos de avaliação da conformidade. 3.3.2. Cláusula de salvaguarda – os poderes especiais do presidente do Conselho de Administração do INFARMED. 3.4. O sistema de vigilância (vide Portaria n.º 196/2004, de 1 de Março: aprova o Regulamento do Sistema Nacional de Vigilância de Dispositivos Médicos). 3.5. Fiscalização e contraordenaçõe

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host’s growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23–28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control

Early diverging insect-pathogenic fungi of the order entomophthorales possess diverse and unique subtilisin-like serine proteases

Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin-associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or very few host species. The extent to which insect-pathogenic fungi in the order Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants (Formica polyctena), respectively. In total 154 SLSP from six fungi in the subphylum Entomophthoromycotina were identified: E. muscae (n = 22), P. formicae (n = 6), B. meristosporus (n = 60), C. thromboides (n = 18), C. coronatus (n = 36), and C. incongruus (n = 12). A unique group of 11 SLSPs was discovered in the genomes of the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus that loosely resembles bacillopeptidase F-like SLSPs. Phylogenetics and protein domain analysis show this class represents a unique group of SLSPs so far only observed among Bacteria, Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and Entomophthoromycotina. This group of SLSPs is missing in the sister fungal lineages of Kickxellomycotina and the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota fungi suggesting interesting gene loss patterns

Draft genome of the fungus-growing termite pathogenic fungus <i>Ophiocordyceps bispora</i> (Ophiocordycipitaceae, Hypocreales, Ascomycota)

This article documents the public availability of genome sequence data and assembled contigs representing the partial draft genome of Ophiocordyceps bispora. As one of the few known pathogens of fungus-farming termites, a draft genome of O. bispora represents the opportunity to further the understanding of disease and resistance in these complex termite societies. With the ongoing attempts to resolve the taxonomy of the Hypocralaean family, more genetic data will also help to shed light on the phylogenetic relationship between sexual and asexual life stages. Next generation sequence data is available from the European Nucleotide Archive (ENA) under accession PRJEB13655; run numbers: ERR1368522, ERR1368523, and ERR1368524. Genome assembly available from ENA under accession numbers: FKNF01000001–FKNF01000302. Gene prediction available as protein fasta, nucleotide fasta and GFF file from Mendeley Data with accession doi:10.17632/r99fd6g3s4.2 (http://dx.doi.org/10.17632/r99fd6g3s4.2)

Knowledge of learning disabilities: the relationship with choice, duty of care and non-aversive approaches

The present study examines the relationship between the knowledge of the diagnostic criteria for a learning disability (based on DSM IV criteria), care practices and experience in health care and social care staff. Responses to a questionnaire were analysed in terms of participants emphasis on: recognizing duty of care; enabling choice; non-aversive and aversive strategies. Results indicated that the knowledge of the criteria for a learning disability was limited, with only I6% of the sample correctly identifying all three criteria. There were no significant differences between the two groups in relation to experience or level of knowledge. No clear cut differences were found between the groups in relation to tendency to emphasize a particular management approach, with the strategies adopted appearing to be influenced by vignettes used in this study. Participants tended to give responses that identified both a recognition of their duty of care to clients and the need to enable choice. Limitations of this study are discussed