Regulation of sexual differentiation initiation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism to explore cellular events owing to rich tools in genetics, molecular biology, cellular biology, and biochemistry. Schizosaccharomyces pombe proliferates continuously when nutrients are abundant but arrests in G1 phase upon depletion of nutrients such as nitrogen and glucose. When cells of opposite mating types are present, cells conjugate, fuse, undergo meiosis, and finally form 4 spores. This sexual differentiation process in S. pombe has been studied extensively. To execute sexual differentiation, the glucose-sensing cAMP-PKA (cyclic adenosine monophosphate-protein kinase A) pathway, nitrogen-sensing TOR (target of rapamycin) pathway, and SAPK (stress-activating protein kinase) pathway are crucial, and the MAPK (mitogen-activating protein kinase) cascade is essential for pheromone sensing. These signals regulate ste11 at the transcriptional and translational levels, and Ste11 is modified in multiple ways. This review summarizes the initiation of sexual differentiation in S. pombe based on results I have helped to obtain, including the work of many excellent researchers.

Spores of arbuscular mycorrhizal fungi host surprisingly diverse communities of endobacteria.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant root symbionts, which can house two endobacteria: Ca. Moeniiplasma glomeromycotorum (CaMg) and Ca. Glomeribacter gigasporarum (CaGg). However, little is known about their distribution and population structure in natural AMF populations and whether AMF can harbour other endobacteria. We isolated AMF from two environments and conducted detailed analyses of endobacterial communities associated with surface-sterilised AMF spores. Consistent with the previous reports, we found that CaMg were extremely abundant (80%) and CaGg were extremely rare (2%) in both environments. Unexpectedly, we discovered an additional and previously unknown level of bacterial diversity within AMF spores, which extended beyond the known endosymbionts, with bacteria belonging to 10 other phyla detected across our spore data set. Detailed analysis revealed that: CaGg were not limited in distribution to the Gigasporaceae family of AMF, as previously thought; CaMg population structure was driven by AMF host genotype; and a significant inverse correlation existed between the diversity of CaMg and diversity of all other endobacteria. Based on these data, we generate novel testable hypotheses regarding the function of CaMg in AMF biology by proposing that they might act as conditional mutualists of AMF.

Gamma-irradiated Aspergillus conidia show a growth curve with a reproductive death phase.

In this study, we evaluated the effects of gamma irradiation on the germination of Aspergillus conidia and mycelial growth using microscopy and predictive microbiological modeling methods. A dose of 0.4 kGy reduced the germination rate by 20% compared to the untreated control, indicating interphase death due to the high radiation dose. The number of colonies formed (5.5%) was lower than the germination rate (69%), suggesting that most colonies died after germination. Microscopic observations revealed that mycelial elongation ceased completely in the middle of the growth phase, indicating reproductive death. The growth curves of irradiated conidia exhibited a delayed change in the growth pattern, and a decrease in slope during the early stages of germination and growth at low densities. A modified logistic model, which is a general purpose growth model that allows for the evaluation of subpopulations, was used to fit the experimental growth curves. Dose-dependent waveform changes may reflect the dynamics of the subpopulations during germination and growth. These methods revealed the occurrence of two cell death populations resulting from gamma irradiation of fungal conidia and contribute to the understanding of irradiation-induced cell death in fungi.

Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction.

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

Congo red induces trans-priming to UV-B radiation in Metarhizium robertsii.

Metarhizium spp. is used as a biocontrol agent but is limited because of low tolerance to abiotic stress. Metarhizium robertsii is an excellent study model of fungal pathogenesis in insects, and its tolerance to different stress conditions has been extensively investigated. Priming is the time-limited pre-exposure of an organism to specific stress conditions that increases adaptive response to subsequent exposures. Congo red is a water-soluble azo dye extensively used in stress assays in fungi. It induces morphological changes and weakens the cell wall at sublethal concentrations. Therefore, this chemical agent has been proposed as a stressor to induce priming against other stress conditions in entomopathogenic fungi. This study aimed to evaluate the capacity of Congo red to induce priming in M. robertsii. Conidia were grown on potato dextrose agar with or without Congo red.The tolerance of conidia produced from mycelia grown in these three conditions was evaluated against stress conditions, including osmotic, oxidative, heat, and UV-B radiation. Conidia produced on medium supplemented with Congo red were significantly more tolerant to UV-B radiation but not to the other stress conditions assayed. Our results suggest that Congo red confers trans-priming to UV-B radiation but not for heat, oxidative, or osmotic stress.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Spore traits mediate disturbance effects on arbuscular mycorrhizal fungal community composition and mutualisms.

Trait-based approaches in ecology are powerful tools for understanding how organisms interact with their environment. These approaches show particular promise in disturbance and community ecology contexts for understanding how disturbances like prescribed fire and bison grazing influence interactions between mutualists like arbuscular mycorrhizal (AM) fungi and their plant hosts. In this work we examined how disturbance effects on AM fungal spore community composition and mutualisms were mediated by selection for specific functional spore traits at both the species and community level. We tested these questions by analyzing AM fungal spore communities and traits from a frequently burned and grazed (bison) tallgrass prairie system and using these spores to inoculate a plant growth response experiment. Selection for darker, pigmented AM fungal spores, changes in the abundance and volume of individual AM fungal taxa, and altered sporulation, were indicators of fire and grazing effects on AM fungal community composition. Disturbance associated changes in AM fungal community composition were then correlated with altered growth responses of Schizachyrium scoparium grass. Our work shows that utilization of trait-based approaches in ecology can clarify the mechanisms that underly belowground responses to disturbance, and provide a useful framework for understanding interactions between organisms and their environment.

Differential effects of exposure to toxic or nontoxic mold spores on brain inflammation and Morris water maze performance.

People who live or work in moldy buildings often complain of "brain fog" that interferes with cognitive performance. Until recently, there was no published research on the effects of controlled exposure to mold stimuli on cognitive function or an obvious mechanism of action, fueling controversy over these claims. The constellation of health problems reported by mold-exposed individuals (respiratory issues, fatigue, pain, anxiety, depression, and cognitive deficits) correspond to those caused by innate immune activation following exposure to bacterial or viral stimuli. To determine if mold-induced innate immune activation might cause cognitive issues, we quantified the effects of both toxic and nontoxic mold on brain immune activation and spatial memory in the Morris water maze. We intranasally administered either 1) intact, toxic Stachybotrys chartarum spores; 2) ethanol-extracted, nontoxic Stachybotrys chartarum spores; or 3) control saline vehicle to mice. Inhalation of nontoxic spores caused significant deficits in the test of long-term memory of platform location, while not affecting short-term memory. Inhalation of toxic spores increased motivation to reach the platform. Interestingly, in both groups of mold-exposed males, numbers of interleukin-1ß-immunoreactive cells in many areas of the hippocampus significantly correlated with latency to find the platform, path length, and swimming speed during training, but not during testing for long-term memory. These data add to our prior evidence that mold inhalation can interfere with cognitive processing in different ways depending on the task, and that brain inflammation is significantly correlated with changes in behavior.

Use of X-ray irradiation for inactivation of Aspergillus in cannabis flower.

California cannabis regulations require testing for four pathogenic species of Aspergillus-A. niger, A. flavus, A. fumigatus and A. terreus in cannabis flower and cannabis inhalable products. These four pathogenic species of Aspergillus are important human pathogens and their presence in cannabis flower and cannabis products may pose a threat to human health. In this study, we examined the potential of X-ray irradiation for inactivation of cannabis flower contaminated with any of the four pathogenic species of Aspergillus. We determined that X-ray irradiation at a dose of 2.5 kGy is capable of rendering Aspergillus cells non-viable at low (102 spores/g dried flower), medium (103 spores/g dried flower) and high (104 spores/g dried flower) levels of inoculation. We also showed that X-ray treatment of cannabis flower did not significantly alter the cannabinoid or the terpene profiles of the flower samples. Therefore, X-ray irradiation may be a feasible method for Aspergillus decontamination of cannabis flower. More work is required to determine the consumer safety of irradiated cannabis flower and cannabis products.

Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well-characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity.

Characterization of two infection-induced transcription factors of Magnaporthe oryzae reveals their roles in regulating early infection and effector expression.

The initial stage of rice blast fungus, Magnaporthe oryzae, infection, before 36 h postinoculation, is a critical timespan for deploying pathogen effectors to overcome the host's defences and ultimately cause the disease. However, how this process is regulated at the transcription level remains largely unknown. This study functionally characterized two M. oryzae Early Infection-induced Transcription Factor genes (MOEITF1 and MOEITF2) and analysed their roles in this process. Target gene deletion and mutant phenotype analysis showed that the mutants Δmoeitf1 and Δmoeitf2 were only defective for infection growth but not for vegetative growth, asexual/sexual sporulation, conidial germination, and appressoria formation. Gene expression analysis of 30 putative effectors revealed that most effector genes were down-regulated in mutants, implying a potential regulation by the transcription factors. Artificial overexpression of two severely down-regulated effectors, T1REP and T2REP, in the mutants partially restored the pathogenicity of Δmoeitf1 and Δmoeitf2, respectively, indicating that these are directly regulated. Yeast one-hybrid assay and electrophoretic mobility shift assay indicated that Moeitf1 specifically bound the T1REP promoter and Moeitf2 specifically bound the T2REP promoter. Both T1REP and T2REP were predicted to be secreted during infection, and the mutants of T2REP were severely reduced in pathogenicity. Our results indicate crucial roles for the fungal-specific Moeitf1 and Moeitf2 transcription factors in regulating an essential step in M. oryzae early establishment after penetrating rice epidermal cells, highlighting these as possible targets for disease control.

Nitrogen fertilisation disrupts the temporal dynamics of arbuscular mycorrhizal fungal hyphae but not spore density and community composition in a wheat field.

Elucidating the temporal dynamics of arbuscular mycorrhizal (AM) fungi is critical for understanding their functions. Furthermore, research investigating the temporal dynamics of AM fungi in response to agricultural practices remains in its infancy. We investigated the effect of nitrogen fertilisation and watering reduction on the temporal dynamics of AM fungi, across the lifespan of wheat. Nitrogen fertilisation decreased AM fungal spore density (SD), extraradical hyphal density (ERHD), and intraradical colonisation rate (IRCR) in both watering conditions. Nitrogen fertilisation affected AM fungal community composition in soil but not in roots, regardless of watering conditions. The temporal analysis revealed that AM fungal ERHD and IRCR were higher under conventional watering and lower under reduced watering in March than in other growth stages at low (≤ 70 kg N ha-1 yr-1 ) but not at high (≥ 140) nitrogen fertilisation levels. AM fungal SD was lower in June than in other growth stages and community composition varied with plant development at all nitrogen fertilisation levels, regardless of watering conditions. This study demonstrates that high nitrogen fertilisation levels disrupt the temporal dynamics of AM fungal hyphal growth but not sporulation and community composition.

The MoPah1 phosphatidate phosphatase is involved in lipid metabolism, development, and pathogenesis in Magnaporthe oryzae.

As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up-regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium-like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection-related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast.

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane-nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ-tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo-like kinase Cdc5 and the meiosis-specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase-promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking-activation mechanism ensures that only single-copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II-specific processes.

AoATG5 plays pleiotropic roles in vegetative growth, cell nucleus development, conidiation, and virulence in the nematode-trapping fungus Arthrobotrys oligospora.

Autophagy is an evolutionarily conserved process in eukaryotes, which is regulated by autophagy-related genes (ATGs). Arthrobotrys oligospora is a representative species of nematode-trapping (NT) fungi that can produce special traps for nematode predation. To elucidate the biological roles of autophagy in NT fungi, we characterized an orthologous Atg protein, AoAtg5, in A. oligospora. We found that AoATG5 deletion causes a significant reduction in vegetative growth and conidiation, and that the transcript levels of several sporulation-related genes were significantly downregulated during sporulation stage. In addition, the cell nuclei were significantly reduced in the ΔAoATG5 mutant, and the transcripts of several genes involved in DNA biosynthesis, repair, and ligation were significantly upregulated. In ΔAoATG5 mutants, the autophagic process was significantly impaired, and trap formation and nematocidal activity were significantly decreased. Comparative transcriptome analysis results showed that AoAtg5 is involved in the regulation of multiple cellular processes, such as autophagy, nitrogen metabolism, DNA biosynthesis and repair, and vesicular transport. In summary, our results suggest that AoAtg5 is essential for autophagy and significantly contributes to vegetative growth, cell nucleus development, sporulation, trap formation, and pathogenicity in A. oligospora, thus providing a basis for future studies focusing on related mechanisms of autophagy in NT fungi.

Courtship Ritual of Male and Female Nuclei during Fertilization in Neurospora crassa.

Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants, and many eukaryotic microorganisms, such as fungi. However, the sequential cell biology of fertilization and the associated nuclear dynamics after plasmogamy are poorly understood in filamentous fungi. Using histone-fluorescent parental isolates, we tracked male and female nuclei during fertilization in the model ascomycete Neurospora crassa using live-cell imaging. This study unravels the behavior of trichogyne resident female nuclei and the extraordinary manner in which male nuclei migrate up the trichogyne to the protoperithecium. Our observations raise new fundamental questions about the modus operandi of nucleus movements during sexual reproduction, male and female nuclear identity, guidance of nuclei within the trichogyne and, unexpectedly, the avoidance of "polyspermy" in fungi. The spatiotemporal dynamics of male nuclei within the trichogyne following plasmogamy are also described, where the speed and the deformation of male nuclei are of the most dramatic observed to date in a living organism. IMPORTANCE Using live-cell fluorescence imaging, for the first time we have observed live male and female nuclei during sexual reproduction in the model fungus Neurospora crassa. This study reveals the specific behavior of resident female nuclei within the trichogyne (the female organ) after fertilization and the extraordinary manner in which male nuclei migrate across the trichogyne toward their final destination, the protoperithecium, where karyogamy takes place. Importantly, the speed and deformation of male nuclei were found to be among the most dramatic ever observed in a living organism. Furthermore, we observed that entry of male nuclei into protoperithecia may block the entry of other male nuclei, suggesting that a process analogous to polyspermy avoidance could exist in fungi. Our live-cell imaging approach opens new opportunities for novel research on cell-signaling during sexual reproduction in fungi and, on a broader scale, nuclear dynamics in eukaryotes.

Epidermal galactose spurs chytrid virulence and predicts amphibian colonization.

The chytrid fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans cause the skin disease chytridiomycosis in amphibians, which is driving a substantial proportion of an entire vertebrate class to extinction. Mitigation of its impact is largely unsuccessful and requires a thorough understanding of the mechanisms underpinning the disease ecology. By identifying skin factors that mediate key events during the early interaction with B. salamandrivorans zoospores, we discovered a marker for host colonization. Amphibian skin associated beta-galactose mediated fungal chemotaxis and adhesion to the skin and initiated a virulent fungal response. Fungal colonization correlated with the skin glycosylation pattern, with cutaneous galactose content effectively predicting variation in host susceptibility to fungal colonization between amphibian species. Ontogenetic galactose patterns correlated with low level and asymptomatic infections in salamander larvae that were carried over through metamorphosis, resulting in juvenile mortality. Pronounced variation of galactose content within some, but not all species, may promote the selection for more colonization resistant host lineages, opening new avenues for disease mitigation.

Aeciospore ejection in the rust pathogen Puccinia graminis is driven by moisture ingress.

Fungi have evolved an array of spore discharge and dispersal processes. Here, we developed a theoretical model that explains the ejection mechanics of aeciospore liberation in the stem rust pathogen Puccinia graminis. Aeciospores are released from cluster cups formed on its Berberis host, spreading early-season inoculum into neighboring small-grain crops. Our model illustrates that during dew or rainfall, changes in aeciospore turgidity exerts substantial force on neighboring aeciospores in cluster cups whilst gaps between spores become perfused with water. This perfusion coats aeciospores with a lubrication film that facilitates expulsion, with single aeciospores reaching speeds of 0.053 to 0.754 m·s-1. We also used aeciospore source strength estimates to simulate the aeciospore dispersal gradient and incorporated this into a publicly available web interface. This aids farmers and legislators to assess current local risk of dispersal and facilitates development of sophisticated epidemiological models to potentially curtail stem rust epidemics originating on Berberis.

The pathological effects of a Nosema ceranae infection in the giant honey bee, Apis dorsata Fabricius, 1793.

Nosema ceranae is an intracellular microsporidian pathogen that lives in the midgut ventricular cells of all known honey bee Apis species. We suspect that N. ceranae may also cause energetic stress in the giant honey bee because this parasite is known to disrupt nutrient absorption resulting in energetic stress in the honey bee species Apis mellifera. To understand how N. ceranae impacts the energetic stress of the giant honey bee, A. dorsata, we measured the hemolymph trehalose levels of experimentally infected giant honey bees on days three, five, seven, and fourteen post infection (p.i.). We also measured the hypopharyngeal gland protein content, the total midgut proteolytic enzyme activity, honey bee survival, infection ratio, and spore loads comparing infected and uninfected honey bees across the same time frame. Nosema ceranae-infected honey bees had significantly lowered survival, trehalose levels, hypopharyngeal gland protein content, and midgut proteolytic enzyme activity. We found an increasing level of parasitic loads and infection ratio of N. ceranae-infected bees after inoculation. Collectively, our results suggest that the giant honey bee suffers from energetic stress and limited nutrient absorption from a N. ceranae infection, which results in lowered survival in comparison to uninfected honey bees. Our findings highlight that other honey bee species besides A. mellifera are susceptible to microsporidian pathogens that they harbor, which results in negative effects on health and survival. Therefore, these pathogens might be transmitted at a community level, in the natural environment, resulting in negative health effects of multiple honey bee species.

An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2).

Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host-pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL-1) was better than other two M. anisopliae (134.59 µg mL-1) and B. bassiana (128.65 µg mL-1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL-1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL-1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL-1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.