The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

Microbiome changes in Akanthomyces attenuatus JEF-147-infected two-spotted spider mites.

The two-spotted spider mite (Tetranychus urticae Koch) is an agriculturally serious polyphagous pest that has acquired strong resistance against acaricides because of its short life cycle and continuous exposure to acaricides. As an alternative, mite-pathogenic fungi with different modes of action could be used to control the mites. The spider mite has symbiotic microorganisms that could be involved in the physiological and ecological adaptations to biotic stresses. In this study, mite-pathogenic fungi were used to control female adults, and the microbiomes changes in the fungus-infected mites were analyzed. The acaricidal activity of 77 fungal isolates was tested, and Akanthomyces attenuatus JEF-147 exhibited the highest acaricidal activity. Subsequently a dose-response assay and morphological characterization was undertaken For microbiome analysis in female adults infected with A. attenuatus JEF-147, 16S rDNA and ITS1 were sequenced using Illumina Miseq. Infected mite showed a higher Shannon index in bacterial diversity but lower index in fungal diversity. In beta diversity using principal component analysis, JEF-147-treated mites were significantly different from non-treated controls in both bacteria and fungi. Particularly in bacterial abundance, arthropod defense-related Rickettsia increased, but arthropod reproduction-associated Wolbachia decreased. The change in major bacterial abundance in the infected mites could be explained by a trade-off between reproduction and immunity against the early stage of fungal attack. In fungal abundance, Akanthomyces showed up as expected. Foremost, this work reports microbiome changes in a fungus-infected mite and suggests a possible trade-off in mites against fungal pathogens. Future studies will focus on gene-based investigations related to this topic.

Biodegradation of pesticide in agricultural soil employing entomopathogenic fungi: Current state of the art and future perspectives.

Pesticides play a pivotal role in agriculture for the effective production of various crops. The indiscriminate use of pesticides results in the significant bioaccumulation of pesticide residues in vegetables. This situation is beyond the control of consumers and poses a serious health issue for human beings. Occupational exposure to pesticides may occur for farmers, agricultural workers, and industrial producers of pesticides. This occupational exposure primarily causes food and water contamination that gets into humans and environmental pollution. Depending on the toxicity of pesticides, the causes and effects differ in the environment and in human health. The number of criteria used and the method of implementation employed to assess the effect of pesticides on humans and the environment have been increasing, as they may provide characterization of pesticides that are already on the market as well as those that are on the way. The biological control of pests has been increasing nowadays to combat all these effects caused by synthetic pesticides. Myco-biocontrol has received great attention in research because it has no negative impact on humans, the environment, or non-target species. Entomopathogenic fungi are microbes that have the ability to kill insect pests. Fungi also make enzymes like the lytic enzymes, esterase, oxidoreductase, and cytochrome P450, which react with chemical residues in the field and break them down into nontoxic substances. In this review, the authors looked at how entomopathogenic fungi break down insecticides in the environment and how their enzymes break down insecticides on farms.

Evaluation of a guardian plant system to suppress Frankliniella occidentalis (Thysanoptera: Thripidae) in greenhouse ornamentals.

BACKGROUND: Western flower thrips (WFT), Franklinella occidentalis (Pergande), is an economically damaging pest of greenhouse ornamentals. A 'guardian plant system' (GPS) that targeted WFT was evaluated under controlled and commercial greenhouse conditions. This system used mycotized millet grains with the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin applied to soil of potted marigolds, Tagetes patula (L.), combined with the foliar-dwelling predatory mite Neoseiulus (=Amblyseius) cucumeris (Oudemans) in slow-release sachets under controlled greenhouse conditions, and with the addition of a pheromone lure under commercial settings. RESULTS: Significantly fewer WFT and less foliar damage on GPS was observed over the 10 and 12 weeks experimental periods compared to the untreated controls. Predatory mites were maintained up to 10 weeks with one release under controlled greenhouse conditions and 12 weeks with two releases in commercial greenhouses. In commercial greenhouses, greater numbers of WFT were found on marigolds than on crop plants within 1 m of the system. Fungal granules persisted for 12 weeks up to 2.5 × 105 CFU g-1 in the GPS soil. CONCLUSION: The use of biological control agents to suppress WFT within a GPS could be a useful IPM strategy for greenhouse production. The marigold GPS attracted WFT which were suppressed primarily through predation by foliar-dwelling predatory mites and to a lesser extent, infection from conidia produced by the granular fungal formulation in the soil. Further investigations into system deployment and fungal granular application rates and new fungal formulations are suggested to improve system efficacy. © 2023 Society of Chemical Industry.

Detection of viral genes in Metarhizium anisopliae JEF-290-infected longhorned tick, Haemaphysalis longicornis using transcriptome analysis.

Ticks are carriers of viruses that can cause disease in humans and animals. The longhorned ticks (Haemaphysalis longicornis; LHT), for example, mediates the severe fever with thrombocytopenia syndrome virus (SFTSV) in humans, and the population of ticks is growing due to increases in temperature caused by climate change. As ticks carry primarily RNA viruses, there is a need to study the possibility of detecting new viruses through tick virome analysis. In this study, viruses in LHTs collected in Korea were investigated and virus titers in ticks exposed to the entomopathogenic fungus Metarhizium anisopliae JEF-290 were analyzed. Total RNA was extracted from the collected ticks, and short reads were obtained from Illumina sequencing. A total of 50,024 contigs with coding capacity were obtained after de novo assembly of the reads in the metaSPAdes genome assembler. A series of BLAST-based analyses using the GenBank database was performed to screen viral contigs, and three putative virus species were identified from the tick meta-transcriptome, such as Alongshan virus (ALSV), Denso virus and Taggert virus. Measurements of virus-expression levels of infected and non-infected LHTs failed to detect substantial differences in expression levels. However, we suggest that LHT can spread not only SFTSV, but also various other disease-causing viruses over large areas of the world. From the phylogenetic analysis of ALSV glycoproteins, genetic differences in the ALSV could be due to host differences as well as regional differences. Viral metagenome analysis can be used as a tool to manage future outbreaks of disease caused by ticks by detecting unknown viruses.

Pathogenesis and defense mechanism while Beauveria bassiana JEF-410 infects poultry red mite, Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae (Mesostigmata: Dermanyssidae), is a major pest that causes great damage to chicken egg production. In one of our previous studies, the management of red mites using entomopathogenic fungi was evaluated, and the acaricidal fungus Beauveria bassiana JEF-410 was selected for further research. In this study, we tried to elucidate the pathogenesis of B. bassiana JEF-410 and the defense mechanisms of red mites at a transcriptome level. Red mites collected from a chicken farm were treated with B. bassiana JEF-410. When the mortality of infected red mites reached 50%, transcriptome analyses were performed to determine the interaction between B. bassiana JEF-410 and red mites. Uninfected red mites and non-infecting fungus served as controls. In B. bassiana JEF-410, up-regulated gene expression was observed in tryptophan metabolism and secondary metabolite biosynthesis pathways. Genes related to acetyl-CoA synthesis were up-regulated in tryptophan metabolism, suggesting that energy metabolism and stress management were strongly activated. Secondary metabolites associated with fungal up-regulated DEGs were related to the production of substances toxic to insects such as beauvericin and beauveriolide, efflux pump of metabolites, energy production, and resistance to stress. In red mites, physical and immune responses that strengthen the cuticle against fungal infection were highly up-regulated. From these gene expression analyses, we identified essential factors for fungal infection and subsequent defenses of red mites. These results will serve as a strong platform for explaining the interaction between B. bassiana JEF-410 and red mites in the stage of active infection.

Management of overwintering pine sawyer beetle, Monochamus alternatus with colonized Beauveria bassiana ERL836.

Monochamus alternatus is a major forest pest that spreads pine wilt disease in pine trees as a vector of pine wilt nematodes. Chemical insecticides used as fumigants to control overwintering M. alternatus in forests are highly toxic to the environment, so we investigated entomopathogenic fungus Beauveria bassiana ERL836 as an eco-friendly and alternative material to control overwintering M. alternatus. In this work, we evaluated the insecticidal activity of B. bassiana ERL836 against M. alternatus adults, the possibility of fungal colonization on pine tree bark, and finally the control efficacy of fungal pre-treatment on pine tree logs against emerging M. alternatus adults in semi-field and field conditions. M. alternatus adults were killed on the pine tree logs pre-treated with the B. bassiana ERL836. White conidia were observed not only on the surface of the dead adults but also on the pine tree logs, suggesting that the adults were killed by the fungus on the pine. A formulated ERL836 powder treatment on larvae-infested pine logs showed high insecticidal activity against adults, similar to that with the fungal powder suspension treatment, but we demonstrated that using the fungal powder was simpler than using the suspension in field conditions. Even in the field condition, the fungal powder treatment showed high insecticidal activity against M. alternatus adults, which we attribute to its ability to maintain fungal activity for a long time in field conditions by covering the pine tree logs with a film during overwintering. We confirmed that the risk that fungus-infected M. alternatus adults would spread the fungus to other non-target forest insects was low. Thus, even a high-concentration treatment in a specific area is unlikely to transmit the fungus outside that area, so it can be safely used to control this pine wilt nematode vector in forest ecosystems.

Whole-Genome Sequence of Beauveria bassiana JEF-350, a Strain with High Insecticidal Activity against Melon Thrips (Thrips palmi).

The entomopathogenic fungus Beauveria bassiana JEF-350 was isolated from forest soil in South Korea. Here, we report the whole-genome sequence of JEF-350, along with the analyzed genetic information, which can be used to study insecticidal mechanisms and fungal diversity.

Early-Stage Defense Mechanism of the Cotton Aphid Aphis gossypii Against Infection With the Insect-Killing Fungus Beauveria bassiana JEF-544.

Aphis gossypii, commonly known as the cotton aphid, is a widely distributed pest of agricultural crops and acts as a vector for many serious plant viruses. Cotton aphid shows high resistance to chemical insecticides due to rapid rates of genetic diversity as a result of its short life cycle, seasonal migration, and host alteration. As an alternative, entomopathogenic fungi can be used to control cotton aphids in an environmentally sound manner. However, little is known about how cotton aphids respond to fungal infection. In this work, a new Beauveria bassiana strain JEF-544 (Bb JEF-544) was selected and isolated through bioassays with high virulence against cotton aphid. Early response of cotton aphid to Bb JEF-544 infection was analyzed at the transcriptome level. Infected aphids were collected two days after treatment at 25% lethal time (LT25), and total RNA of non-infected and Bb JEF-544-infected aphids was independently subjected to sequencing. Infected aphids showed significant up-regulation of the insect hormone biosynthesis pathway. Bursicon (Burs) and crustacean cardioactive peptide (CCAP) receptors involved in molting along with ecdysone synthesis were also strongly up-regulated in the aphid response to the fungal infection. In the immune response, melanization in the hemocoel was significantly up-regulated, while phagocytosis was less actively transcribed. In conclusion, cotton aphids protect themselves from Bb JEF-544 infection by activating the immune response including melanization and insect molting hormones to shed infected cuticles. In addition to describing the initial stages of Bb JEF-544 infection at the transcriptome level, this work provides potential treatment targets and insight into how fungal isolates can effectively be used to control this serious aphid species.

Ultrastructural analysis of beetle larva cuticles during infection with the entomopathogenic fungus, Beauveria bassiana.

BACKGROUND: Beauveria bassiana is one of the commercially available entomopathogenic fungi (EPF), and a number of isolates with high virulence and broad host spectrum have been used to control agricultural and forest pests. Although the functional importance of genes in EPFs' pathogenesis have been extensively studied, the precise ultrastructural mechanism of the fungal infection, particularly penetration of the host insect cuticles, is not well understood. RESULTS: In this study, we investigated the morphology and ultrastructure of the larval cuticle of the red flour beetle, Tribolium castaneum, after treatment with B. bassiana ERL1170 expressing an enhanced green fluorescent protein (Bb-eGFP). The Bb-eGFP showed high virulence against the larvae, with approximately 90% mortality at 48 h after treatment (HAT) and 100% at 72 HAT under our infection conditions. In these larvae, the regions of the body wall with flexible cuticles, such as the ventral and ventrolateral thorax and abdomen, became darkly melanized, but there was little to no melanization in the rigid dorsal cuticular structures. Confocal microscopy and transmission electron microscopy (TEM) indicated that germinated conidia on the surface of the larval cuticle were evident at 6 HAT, which formed penetration pegs and began to penetrate the several cuticle layers/laminae by 12 HAT. The penetration pegs then developed invading hyphae, some of which passed through the cuticle and reached the epidermal cells by 24 HAT. The larval cuticle was aggressively and extensively disrupted by 48 HAT, and a number of outgrowing hyphae were observed at 72 HAT. CONCLUSIONS: Our results indicate that Bb-eGFP is capable of infection and penetrating T. castaneum larvae shortly after inoculation (~24 HAT) at the body regions with apparently flexible and membranous cuticles, such as the ventral intersegmental regions and the ventrolateral pleura. This study provides details on the histopathogenesis of the host cuticle by infection and penetration of EPFs, which can facilitate the management of insect pests. © 2022 Society of Chemical Industry.

Effect of storage conditions on the shelf-life extension of fungus-colonized substrates based on Metarhizium anisopliae using modified atmosphere packaging.

Metarhizium anisopliae is a promising alternative to chemical pesticides against pine wilt disease caused by Bursaphelenchus xylophilus. Herein, we investigated the efficacy of modified atmosphere packaging (MAP) to prolong the shelf-life of the M. anisopliae conidia. The effects of various conditions on its stability were also examined. M. anisopliae-inoculated millet grains were treated in a MAP system with different packaging materials (polypropylene, PP; polyethylene terephthalate, PET; ethylene vinyl alcohol, EVOH), gas compositions (high CO2 atmosphere, ≈ 90%; high O2 atmosphere, > 95%; high N2 atmosphere, > 95%; 30% CO2 + 70% N2; 50% CO2 + 50% N2; 70% CO2 + 30% N2), and storage temperatures (4 and 25 °C). Results revealed EVOH film as the best for the preservation of gases at all concentrations for 28 days. MAP treatment in the high-barrier EVOH film under an atmosphere of 30% CO2 + 70% N2 achieved 80.5% viability of dried conidia (7.4% moisture content), with 44.2-64.9% viability recorded with the other treatments. Cold storage for technical concentrates formulation promoted extension of shelf-life of MAP-treated conidia. These results imply that MAP under optimized conditions could enhance the shelf-life of fungus-based biopesticides in fungus-colonized substrates formulations.

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Interactive Gene Expression Between Metarhizium anisopliae JEF-290 and Longhorned Tick Haemaphysalis longicornis at Early Stage of Infection.

The longhorned tick, Haemaphysalis longicornis (Acari: Ixodidae), is a hard tick and a vector for severe fever with thrombocytopenia syndrome (SFTS) virus. The number of patients infected with SFTS is rapidly increasing. Recently, the invertebrate pathogen Metarhizium anisopliae JEF-290 was reported to be useful to control the tick as an alternative to chemical acaricides, which are not easily applicable in human living areas where the tick is widely spread. In this study, we analyzed how the tick and the fungal pathogen interact at the transcriptional level. Field-collected tick nymphs were treated with JEF-290 conidia at 1 × 108 conidia/ml. In the early stage of infection with 2.5% mortality, the infected ticks were subjected to RNA sequencing, and non-infected ticks and fungal masses served as controls. Fungus and tick genes were mostly up-regulated at the early stage of infection. In the gene set enrichment analysis of the infecting fungus, catabolic processes that included lipids, phospholipids, and detoxification processes, the response to oxidative stress, and toxic substances were significantly up-regulated. In this fungal up-regulation, various lipase, antioxidant enzyme, and hydrolase genes were highly transcribed. The gene set enrichment analysis of the infected tick showed that many peptide synthesis processes including translation, peptide metabolism, ribonucleotide metabolism, and energy production processes that included ATP generation and ADP metabolism were significantly up-regulated. Structurally, mitochondria and ribosome subunit genes in ticks were highly transcribed to upregulate these processes. Together these results indicate that JEF-290 initiates process that infects the tick while the tick actively defends against the fungal attack. This work provides background to improve our understanding of the early stage of fungal infection in longhorned tick.

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA.

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl2 conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD630 = 0.2) compared to the control culture, Bb-egfp#3 only (OD630 = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.

Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana.

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Beauveria bassiana ERL836 and JEF-007 with similar virulence show different gene expression when interacting with cuticles of western flower thrips, Frankniella occidentalis.

BACKGROUND: Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection. RESULTS: The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836. CONCLUSION: The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

Evaluation of spray applications of Metarhizium anisopliae, Metarhizium brunneum and Beauveria bassiana against larval winter ticks, Dermacentor albipictus.

Dermacentor albipictus (Acari: Ixodidae), the winter tick, is a one-host tick that parasitizes large ungulates. They can dramatically affect moose, Alces alces (Artiodactyla: Cervidae), causing significant physiological and metabolic stress and mortality among heavily parasitized individuals. Entomopathogenic fungi in the genera Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria (Hypocreales: Cordycipitaceae) are promising tick biological control agents. We examined the pathogenicity of experimental and commercially formulated isolates of M. anisopliae, M. brunneum and B. bassiana sprayed at concentrations of 106, 107 and 108 conidia/mL against the larval stage of D. albipictus and assessed the efficacy of spraying the commercial product Met52®EC, containing M. brunneum, strain F52, under laboratory conditions. Results showed larval D. albipictus mortality was significantly higher and occurred earlier when treated with M. anisopliae and M. brunneum isolates compared to B. bassiana at 106, 107 and 108 conidia/mL. Mortality was observed as early as 3 days in the M. anisopliae and M. brunneum treatments and after 6 days in the B. bassiana treatments. After 21 days, larval mortality ranged from 74-99% when ticks were treated with M. anisopliae and M. brunneum isolates at 106, 107 and 108 and conidia/mL. In contrast, mortality of ticks treated with B. bassiana ranged from 30 to 64%. When larvae were treated with the commercial product Met52, mortality was ~ 45% after 3 days and ~ 96% after 9 days. These results demonstrate the effectiveness of M. anisopliae and M. brunneum against D. albipictus.

Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus).

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.