A Drosophila melanogaster model shows that fast growing Metarhizium species are the deadliest despite eliciting a strong immune response.

We used Drosophila melanogaster to investigate how differences between Metarhizium species in growth rate and mechanisms of pathogenesis influence the outcome of infection. We found that the most rapid germinators and growers in vitro and on fly cuticle were the fastest killers, suggesting that pre-penetration competence is key to Metarhizium success. Virulent strains also induced the largest immune response, which did not depend on profuse growth within hosts as virulent toxin-producing strains only proliferated post-mortem while slow-killing strains that were specialized to other insects grew profusely pre-mortem. Metarhizium strains have apparently evolved resistance to widely distributed defenses such as the defensin Toll product drosomycin, but they were inhibited by Bomanins only found in Drosophila spp. Disrupting a gene (Dif), that mediates Toll immunity has little impact on the lethality of most Metarhizium strains (an exception being the early diverged M. frigidum and another insect pathogen Beauveria bassiana). However, disrupting the sensor of fungal proteases (Persephone) allowed rapid proliferation of strains within hosts (with the exception of M. album), and flies succumbed rapidly. Persephone also mediates gender differences in immune responses that determine whether male or female flies die sooner. We conclude that some strain differences in growth within hosts depend on immune-mediated interactions but intrinsic differences in pathogenic mechanisms are more important. Thus, Drosophila varies greatly in tolerance to different Metarhizium strains, in part because some of them produce toxins. Our results further develop D. melanogaster as a tractable model system for understanding insect-Metarhizium interactions.

Bioremediation of mercury-polluted soil and water by the plant symbiotic fungus Metarhizium robertsii.

Fungi are central to every terrestrial and many aquatic ecosystems, but the mechanisms underlying fungal tolerance to mercury, a global pollutant, remain unknown. Here, we show that the plant symbiotic fungus Metarhizium robertsii degrades methylmercury and reduces divalent mercury, decreasing mercury accumulation in plants and greatly increasing their growth in contaminated soils. M. robertsii does this by demethylating methylmercury via a methylmercury demethylase (MMD) and using a mercury ion reductase (MIR) to reduce divalent mercury to volatile elemental mercury. M. robertsii can also remove methylmercury and divalent mercury from fresh and sea water even in the absence of added nutrients. Overexpression of MMD and MIR significantly improved the ability of M. robertsii to bioremediate soil and water contaminated with methylmercury and divalent mercury. MIR homologs, and thereby divalent mercury tolerance, are widespread in fungi. In contrast, MMD homologs were patchily distributed among the few plant associates and soil fungi that were also able to demethylate methylmercury. Phylogenetic analysis suggests that fungi could have acquired methylmercury demethylase genes from bacteria via two independent horizontal gene transfer events. Heterologous expression of MMD in fungi that lack MMD homologs enabled them to demethylate methylmercury. Our work reveals the mechanisms underlying mercury tolerance in fungi, and may provide a cheap and environmentally friendly means of cleaning up mercury pollution.

Metarhizium: an opportunistic middleman for multitrophic lifestyles.

Metarhizium spp. mediate multiple interactions that are usually positive with respect to their long-term plant environment, and negative with respect to short-lived hosts. In particular, their ability to kill a wide range of insects maximizes protection to the plants and provides a resource of nitrogen that the fungus trades with the plant for carbon. Here, we highlight emerging concepts underlying Metarhizium-plant-insect interactions. Experiments on model systems have provided detailed mechanistic knowledge of how these fungi interact with plants and insects, and a greater understanding of the evolutionary forces driving these interactions. However, further integration of studies at the ecological and mechanistic level is needed to evaluate the importance of Metarhizium's multitrophic interactions to the structuring of natural communities.

Insects and their pathogens in a changing climate.

The complex nature of climate change-mediated multitrophic interaction is an underexplored area, but has the potential to dramatically shift transmission and distribution of many insects and their pathogens, placing some populations closer to the brink of extinction. However, for individual insect-pathogen interactions climate change will have complicated hard-to-anticipate impacts. Thus, both pathogen virulence and insect host immunity are intrinsically linked with generalized stress responses, and in both pathogen and host have extensive trade-offs with nutrition (e.g., host plant quality), growth and reproduction. Potentially alleviating or exasperating these impacts, some pathogens and hosts respond genetically and rapidly to environmental shifts. This review identifies many areas for future research including a particular need to identify how altered global warming interacts with other environmental changes and stressors, and how consistent these impacts are across pathogens and hosts. With that achieved we would be closer to producing an overarching framework to integrate knowledge on all environmental interplay and infectious disease events.

Metarhizium: jack of all trades, master of many.

The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common 'specialist generalist' species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host-pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.

Genetic variation for resistance to the specific fly pathogen Entomophthora muscae.

We found substantial variation in resistance to the fly-specific pathogen Entomophthora muscae 'Berkeley' (Entomophthoromycota), in 20 lines from the Drosophila melanogaster Genetic Reference Panel (DGRP). Resistance to E. muscae is positively (r = 0.55) correlated with resistance to the broad host range ascomycete entomopathogen Metarhizium anisopliae (Ma549), indicative of generalist (non-specific) defenses. Most of the lines showing above average resistance to Ma549 showed cross-resistance to E. muscae. However, lines that succumbed quickly to Ma549 exhibited the full range of resistance to E. muscae. This suggests fly populations differ in E. muscae-specific resistance mechanisms as well as generic defences effective against both Ma549 and E. muscae. We looked for trade-offs that could account for inter-line variation, but increases (decreases) in disease resistance to E. muscae are not consistently associated with increases (decreases) of resistance to oxidative stress, starvation stress and sleep indices. That these pathogens are dynamic agents of selection on hosts is reflected in this genetic variation for resistance in lines derived from wild populations.

Going gentle into that pathogen-induced goodnight.

A diverse set of pathogens have evolved extended phenotypes that manipulate the moribund behavior of their various insect hosts. By elevating host positioning at death, a phenomenon called "summit disease", these pathogens have been shown to have higher fitness. Though a few summit disease systems have been intensively characterized, in particular the Ophiocordyceps-ant system, summit diseases lack an overarching theory for the underlying mechanisms of this complex behavioral manipulation. In this article, we combine the gamut of summiting systems into a cohesive framework: we propose two types of summit disease (juvenile and adult), which both exploit natural insect behaviors during periods of quiescence. We place this framework in the context of available literature and propose investigations that follow from this comprehensive understanding of summit disease in insects.

Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso.

Malaria control efforts require implementation of new technologies that manage insecticide resistance. Metarhizium pingshaense provides an effective, mosquito-specific delivery system for potent insect-selective toxins. A semifield trial in a MosquitoSphere (a contained, near-natural environment) in Soumousso, a region of Burkina Faso where malaria is endemic, confirmed that the expression of an insect-specific toxin (Hybrid) increased fungal lethality and the likelihood that insecticide-resistant mosquitoes would be eliminated from a site. Also, as Hybrid-expressing M. pingshaense is effective at very low spore doses, its efficacy lasted longer than that of the unmodified Metarhizium Deployment of transgenic Metarhizium against mosquitoes could (subject to appropriate registration) be rapid, with products that could synergistically integrate with existing chemical control strategies to avert insecticide resistance.

Transgenic Metarhizium pingshaense synergistically ameliorates pyrethroid-resistance in wild-caught, malaria-vector mosquitoes.

Transgenic Metarhizium pingshaense expressing the spider neurotoxin Hybrid (Met-Hybrid) kill mosquitoes faster and at lower spore doses than wild-type strains. In this study, we demonstrate that this approach dovetails with the cornerstone of current malaria control: pyrethroid-insecticides, which are the cornerstone of current malaria control. We used World Health Organization (WHO) tubes, to compare the impact on insecticide resistance of Met-Hybrid with red fluorescent M. pingshaense (Met-RFP), used as a proxy for the wild-type fungus. Insecticides killed less than 20% of Anopheles coluzzii and Anopheles gambiae s.s. mosquitoes collected in a malaria endemic region of Burkina Faso where pyrethroid use is common. Seven days post-infection, mortality for insecticide-sensitive and resistant mosquitoes averaged 94% with Met-Hybrid and 64% with Met-RFP, with LT80 values of 5.32±0.199 days and 7.76±0.183 days, respectively. Eighty nine percent of insecticide-resistant mosquitoes exposed to permethrin five days post-infection with Met-Hybrid died within 24 hours: only 22% died from Met-Hybrid alone over this 24-hour period. Compared to Met-RFP, Met-Hybrid also significantly reduced flight capacity of mosquitoes 3 to 5 days post-infection. Based on WHOPES phase I laboratory susceptibility bioassays, transgenic Met-Hybrid provides effective biological control for adult African malaria vectors that may be used to synergistically manage insecticide resistance with current methods.

Native entomopathogenic Metarhizium spp. from Burkina Faso and their virulence against the malaria vector Anopheles coluzzii and non-target insects.

BACKGROUND: Genetically enhanced Metarhizium pingshaense are being developed for malaria vector control in Burkina Faso. However, not much is known about the local prevalence and pathogenicity of this fungus, so we prospected mosquitoes and plant roots (a common habitat for Metarhizium spp.) for entomopathogenic fungi. RESULTS: Our investigations showed that Metarhizium spp. represented between 29-74% of fungi isolated from plant root rhizospheres in diverse collection sites. At low spore dosages (1 × 106 conidia/ml), two mosquito-derived M. pingshaense isolates (Met_S26 and Met_S10) showed greater virulence against Anopheles coluzzii (LT80 of ~7 days) than isolates tested in previous studies (LT80 of ~10 days). In addition, the local isolates did not cause disease in non-target insects (honeybees and cockroaches). CONCLUSIONS: Our work provides promising findings for isolating local Metarhizium strains for application in mosquito biological control and for future transgenic biocontrol strategies in Burkina Faso.

Stress-induced pyruvate accumulation contributes to cross protection in a fungus.

It is commonly observed that microorganisms subjected to a mild stress develop tolerance not only to higher doses of the same stress but also to other stresses - a phenomenon called cross protection. The mechanisms for cross protection have not been fully revealed. Here, we report that heat shock induced cross protection against UV, oxidative and osmotic/salt stress conditions in the cosmopolitan fungus Metarhizium robertsii. Similarly, oxidative and osmotic/salt stresses also induced cross protection against multiple other stresses. We found that oxidative and osmotic/salt stresses produce an accumulation of pyruvate that scavenges stress-induced reactive oxygen species and promotes fungal growth. Thus, stress-induced pyruvate accumulation contributes to cross protection. RNA-seq and qRT-PCR analyses showed that UV, osmotic/salt and oxidative stress conditions decrease the expression level of pyruvate consumption genes in the trichloroacetic acid cycle and fermentation pathways leading to pyruvate accumulation. Our work presents a novel mechanism for cross protection in microorganisms.

Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii.

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5' untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.

Pyruvate Accumulation Is the First Line of Cell Defense against Heat Stress in a Fungus.

Heat tolerance is well known to be key to fungal survival in many habitats, but our mechanistic understanding of how organisms adapt to heat stress is still incomplete. Using Metarhizium robertsii, an emerging model organism for assessing evolutionary processes, we report that pyruvate is in the vanguard of molecules that scavenge heat-induced reactive oxygen species (ROS). We show that, as well as inducing a rapid burst of ROS production, heat stress also downregulates genes for pyruvate consumption. The accumulating pyruvate is the fastest acting of several M. robertsii ROS scavengers, efficiently reducing protein carbonylation, stabilizing mitochondrial membrane potential, and promoting fungal growth. The acetate produced from pyruvate-ROS reactions itself causes acid stress, tolerance to which is regulated by Hog1 mitogen-activated protein kinase. Heat stress also induces pyruvate accumulation in several other fungi, suggesting that scavenging of heat-induced ROS by pyruvate is widespread.IMPORTANCE Heat is a dangerous challenge for most organisms, as it denatures proteins and induces the production of ROS that inactivate proteins, lipid membranes, and DNA. How organisms respond to this stress is not fully understood. Using the experimentally tractable insect pathogen Metarhizium robertsii as a model organism, we show for the first time that heat stress induces pyruvate production and that this functions as the first line of defense against heat-induced ROS. Heat stress also induces rapid pyruvate accumulation in other fungi, suggesting that pyruvate is a common but unappreciated defense against stress.

Metarhizium robertsii produces indole-3-acetic acid, which promotes root growth in Arabidopsis and enhances virulence to insects.

The plant root colonizing insect-pathogenic fungus Metarhizium robertsii has been shown to boost plant growth, but little is known about the responsible mechanisms. Here we show that M. robertsii promotes lateral root growth and root hair development of Arabidopsis seedlings in part through an auxin [indole-3-acetic acid (IAA)]-dependent mechanism. M. robertsii, or its auxin-containing culture filtrate promoted root proliferation, activated IAA-regulated gene expression and rescued the root hair defect of the IAA-deficient rhd6 Arabidopsis mutant. Substrate feeding assays suggest that M. robertsii possesses tryptamine (TAM) and indole-3-acetamide tryptophan (Trp)-dependent auxin biosynthetic pathways. Deletion of Mrtdc impaired M. robertsii IAA production by blocking conversion of Trp to TAM but the reduction was not sufficient to affect plant growth enhancement. We also show that M. robertsii secretes IAA on insect cuticle. ∆Mrtdc produced fewer infection structures and was less virulent to insects than the wild-type, whereas M. robertsii spores harvested from culture media containing IAA were more virulent. Furthermore, exogenous application of IAA increased appressorial formation and virulence. Together, these results suggest that auxins play an important role in the ability of M. robertsii to promote plant growth, and the endogenous pathways for IAA production may also be involved in regulating entomopathogenicity. Auxins were also produced by other Metarhizium species and the endophytic insect pathogen Beauveria bassiana suggesting that interplay between plant- and fungal-derived auxins has important implications for plant-microbe-insect interactions.

Improved efficacy of an arthropod toxin expressing fungus against insecticide-resistant malaria-vector mosquitoes.

The continued success of malaria control efforts requires the development, study and implementation of new technologies that circumvent insecticide resistance. We previously demonstrated that fungal pathogens can provide an effective delivery system for mosquitocidal or malariacidal biomolecules. Here we compared genes from arthropod predators encoding insect specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under control of a hemolymph-specific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA approved Hybrid (Ca++/K+ channel blocker), was studied for pre-lethal effects. These included reduced blood feeding behavior, with almost 100% of insects infected with ~6 spores unable to transmit malaria within 5 days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na+ channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in 5 days as single toxin strains. Our results identify a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

Genome-wide identification of pathogenicity, conidiation and colony sectorization genes in Metarhizium robertsii.

Metarhizium robertsii occupies a wide array of ecological niches and has diverse lifestyle options (saprophyte, insect pathogen and plant symbiont), that renders it an unusually effective model for studying genetic mechanisms for fungal adaptation. Here over 20,000 M. robertsii T-DNA mutants were screened in order to elucidate genetic mechanism by which M. robertsii replicates and persists in diverse niches. About 287 conidiation, colony sectorization or pathogenicity loci, many of which have not been reported in other fungi were identified. By analysing a series of conidial pigmentation mutants, a new fungal pigmentation gene cluster, which contains Mr-Pks1, Mr-EthD and Mlac1 was identified. A conserved conidiation regulatory pathway containing Mr-BrlA, Mr-AbaA and Mr-WetA regulates expression of these pigmentation genes. During conidiation Mr-BlrA up-regulates Mr-AbaA, which in turn controls Mr-WetA. It was found that Hog1-MAPK regulates fungal conidiation by controlling the conidiation regulatory pathway, and that all three pigmentation genes exercise feedback regulation of conidiation. This work provided the foundation for deeper understanding of the genetic processes behind M. robertsii adaptive phenotypes, and advances our insights into conidiation and pigmentation in this fungus.

The Insect Pathogens.

Fungi are the most common disease-causing agents of insects; aside from playing a crucial role in natural ecosystems, insect-killing fungi are being used as alternatives to chemical insecticides and as resources for biotechnology and pharmaceuticals. Some common experimentally tractable genera, such as Metarhizium spp., exemplify genetic diversity and dispersal because they contain numerous intraspecific variants with distinct environmental and insect host ranges. The availability of tools for molecular genetics and multiple sequenced genomes has made these fungi ideal experimental models for answering basic questions on the genetic and genomic processes behind adaptive phenotypes. For example, comparative genomics of entomopathogenic fungi has shown they exhibit diverse reproductive modes that often determine rates and patterns of genome evolution and are linked as cause or effect with pathogenic strategies. Fungal-insect pathogens represent lifestyle adaptations that evolved numerous times, and there are significant differences in host range and pathogenic strategies between the major groups. However, typically, spores landing on the cuticle produce appressoria and infection pegs that breach the cuticle using mechanical pressure and cuticle-degrading enzymes. Once inside the insect body cavity, fungal pathogens face a potent and comprehensively studied immune defense by which the host attempts to eliminate or reduce an infection. The Fungal Kingdom stands alone in the range, extent, and complexity of their manipulation of arthropod behavior. In part, this is because most only sporulate on cadavers, so they must ensure the dying host positions itself to allow efficient transmission.

The genetic basis for variation in resistance to infection in the Drosophila melanogaster genetic reference panel.

Individuals vary extensively in the way they respond to disease but the genetic basis of this variation is not fully understood. We found substantial individual variation in resistance and tolerance to the fungal pathogen Metarhizium anisopliae Ma549 using the Drosophila melanogaster Genetic Reference Panel (DGRP). In addition, we found that host defense to Ma549 was correlated with defense to the bacterium Pseudomonas aeruginosa Pa14, and several previously published DGRP phenotypes including oxidative stress sensitivity, starvation stress resistance, hemolymph glucose levels, and sleep indices. We identified polymorphisms associated with differences between lines in both their mean survival times and microenvironmental plasticity, suggesting that lines differ in their ability to adapt to variable pathogen exposures. The majority of polymorphisms increasing resistance to Ma549 were sex biased, located in non-coding regions, had moderately large effect and were rare, suggesting that there is a general cost to defense. Nevertheless, host defense was not negatively correlated with overall longevity and fecundity. In contrast to Ma549, minor alleles were concentrated in the most Pa14-susceptible as well as the most Pa14-resistant lines. A pathway based analysis revealed a network of Pa14 and Ma549-resistance genes that are functionally connected through processes that encompass phagocytosis and engulfment, cell mobility, intermediary metabolism, protein phosphorylation, axon guidance, response to DNA damage, and drug metabolism. Functional testing with insertional mutagenesis lines indicates that 12/13 candidate genes tested influence susceptibility to Ma549. Many candidate genes have homologs identified in studies of human disease, suggesting that genes affecting variation in susceptibility are conserved across species.

Identification of Drosophila Mutants Affecting Defense to an Entomopathogenic Fungus.

Fungi cause the majority of insect disease. However, to date attempts to model host-fungal interactions with Drosophila have focused on opportunistic human pathogens. Here, we performed a screen of 2,613 mutant Drosophila lines to identify host genes affecting susceptibility to the natural insect pathogen Metarhizium anisopliae (Ma549). Overall, 241 (9.22%) mutant lines had altered resistance to Ma549. Life spans ranged from 3.0 to 6.2 days, with females being more susceptible than males in all lines. Speed of kill correlated with within-host growth and onset of sporulation, but total spore production is decoupled from host genotypes. Results showed that mutations affected the ability of Drosophila to restrain rather than tolerate infections and suggested trade-offs between antifungal and antibacterial genes affecting cuticle and gut structural barriers. Approximately, 13% of mutations where in genes previously associated with host pathogen interactions. These encoded fast-acting immune responses including coagulation, phagocytosis, encapsulation and melanization but not the slow-response induction of anti-fungal peptides. The non-immune genes impact a wide variety of biological functions, including behavioral traits. Many have human orthologs already implicated in human disorders; while others were mutations in protein and non-protein coding genes for which disease resistance was the first biological annotation.