Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties. Aside from playing a crucial role in natural ecosystems, entomopathogenic fungi are being developed as environmentally friendly alternatives for the control of insect pests. We conducted the first genomic study of two of the best characterized entomopathogens, Metarhizium anisopliae and M. acridum. M. anisopliae is a ubiquitous pathogen of >200 insect species and a plant growth promoting colonizer of rhizospheres. M. acridum is a specific pathogen of locusts. Important findings of this study included: 1) Both M. anisopliae and M. acridum have a very large number of genes encoding secreted proteins, and many of these play roles in fungus-insect interactions. 2) M. anisopliae has more genes than M. acridum, which may be associated with adaptation to multiple insect hosts. 3) Unlike M. acridum, the M. anisopliae genome contains many more transposase genes and shows no evidence of repeat-induced point mutations. The lack of repeat-induced mutations may have allowed the lineage-specific gene duplications that have contributed to its adaptability. 4) High-throughput transcriptomics identified the strategies by which these fungi overcome their insect hosts and achieve specificity. These genome sequences will provide the basis for a comprehensive understanding of fungal–plant–insect interactions and will contribute to our understanding of fungal evolution and ecology.

Chitin is an important component of the fungal cell wall with a family of chitin synthases mediating its synthesis. Here, we report on the genetic characterization of the full suite of seven chitin synthases (MaChsI-VII) identified in the insect pathogenic fungus, Metarhizium acridum. Aberrant distribution of chitin was most evident in targeted gene knockouts of MaChsV and MaChsVII. Mutants of MaChsI, MaChsIII, MaChsIV showed delayed conidial germination, whereas ΔMaChsII and ΔMaChsV mutants germinated more rapidly when compared to the wild-type parent. All MaChs genes impacted conidial yield, but differentially affected stress tolerances. Inactivation of MaChsIII, MaChsV, MaChsVII resulted in cell wall fragility, and ΔMaChsV and ΔMaChsVII mutants showed high sensitivity to Congo red and calcofluor white, suggesting that the three genes are required for cell wall integrity. In addition, ΔMaChsIII and ΔMaChsVII mutants showed the highest sensitivities to heat and UV-B stress. Three of seven chitin synthase genes, MaChsIII, MaChsV, MaChsVII, were found to contribute to fungal virulence. Compared with the wild-type strain, ΔMaChsIII and ΔMaChsV mutants were reduced in virulence by topical inoculation, while the ΔMaChsVII mutant showed more severe virulence defects. Inactivation of MaChsIII, MaChsV, or MaChsVII impaired appressorium formation, affected growth of in insecta produced hyphal bodies, and altered the surface properties of conidia and hyphal bodies, resulting in defects in the ability of the mutant strains to evade insect immune responses. These data provide important links between the physiology of the cell wall and the ability of the fungus to parasitize insects and reveal differential functional consequences of the chitin synthase family in M. acridum growth, stress tolerances, cell wall integrity and virulence. The fungal cell wall is a dynamic and flexible organelle that modulates the interaction of the pathogen with its host and acts as a critical recognition and evasion interface with host defenses. Chitin is a hallmark constituent of the fungal cell wall and all fungi contain multiple chitin synthase (Chs) genes. However, systematic characterization of chitin synthase genes has not yet been reported in entomopathogenic fungi. By using the insect pathogen Metarhizium acridum as a model, we performed a systematic genetic analysis of the seven member Chs family (ChsI-VII) in the insect pathogenic fungus. Construction of strains bearing targeted single gene mutations revealed differential contributions of specific Chs genes to growth, cell wall integrity, and stress responses. In addition, we revealed that three chitin synthase genes MaChsIII, MaChsV and MaChsVII were shown to be important for fungal appressorium formation and evasion of insect cellular and/or humoral defenses, promoting the fungal dimorphic transition to the production of hyphal bodies that occurs within hosts, and ultimately to virulence. These data provide new insights into the roles of Chs genes and chitin as critical components affecting fungal membrane structure and function.

The migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus. RNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively. Analysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme. These data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors.

Fungal diseases are widespread among insects and play a crucial role in naturally regulating insect populations. Mosquitoes, known as vectors for numerous infectious diseases, pose a significant threat to human health. Entomopathogenic fungi (EPF) have emerged as highly promising alternative agents to chemical mosquitocides for controlling mosquitoes at all stages of their life cycle due to their unique infection pathway through direct contact with the insect’s cuticle. In recent years, significant advancements have been made in understanding the infection pathways and pathogenic mechanisms of EPF against mosquitoes. Various strategies involving the use of EPF alone or combinations with other approaches have been employed to target mosquitoes at various developmental stages. Moreover, the application of genetic technologies in fungi has opened up new avenues for enhancing the mosquitocidal efficacy of EPF. This review presents a comprehensive summary of recent advancements in our understanding the pathogenic mechanisms of EPF, their applications in mosquito management, and the combination of EPF with other approaches and employment of transgenic technologies. The biosafety concerns associated with their use and the corresponding approaches are also discussed. The recent progress suggests that EPF have the potential to serve as a future biorational tool for controlling mosquito vectors.

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm’hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm’hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm’hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.