Destruxin A, a non-ribosomal peptide toxin produced by Metarhizium, exhibits potent insecticidal activity by targeting various tissues, organs, and cells of insects. Our previous research has revealed that DA possesses the ability to bind to multiple proteins. In this study, we aimed to identify the most sensitive binding proteins of DA and investigate the physiological processes in which DA regulated. Through RNAi technology, we screened 22 binding proteins of DA in silkworm hemolymph. Among them, the juvenile hormone binding protein (JHBP), a hormone transport protein crucial for growth and development regulation, exhibited the highest sensitivity to DA. Subsequent experiments demonstrated that DA could inhibit the body weight gain of silkworm larvae, accelerate the pupation occurrence, and modulate the content of free juvenile hormone (JH) in the hemolymph. We also observed that DA could induce conformational changes in both the JHBP and the JHBP-JH binding complex. Notably, at low dosage, DA influenced the binding of JHBP to JH, while at high dosage, it irreversibly affected the binding of JHBP to JH. Molecular docking and point-mutant experiments suggested that DA might affect the N-arm of JHBP, which is responsible for JH binding. Additionally, we discovered that JHBP is widely distributed in various tissues of the silkworm, including the epidermis, gut, fat body, Malpighian tubule, gonad, muscle, trachea, and hemocyte. This study provides novel insights into the insecticidal mechanism of DA and enhances our understanding of the pathogenic process of Metarhizium.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

Destruxin A (DA) is a cyclo-hexadepsipeptidic insecticidal mycotoxin isolated from the entomopathogenic fungi, Metarhizium spp. However, its mode of action is unknown. In this study, we isolated 149 candidate DA-binding proteins by drug affinity response target stability, and determined the interactions of 80 canditates with DA in vitro by surface plasmon resonance. The affinity coefficients (KD) ranged from 24 to 469 μM. Binding proteins were functionally diverse and included cytoskeletal components and cell motility, protein transcription and translation pathways, ubiquitin dependent protein metabolic processes, nucleus pore entry and exit, and endoplasmic reticulum vesicle transport and etc. Electron microscopy revealed that DA damaged the cytoskeleton and multiple organelles, disrupted cell adhesion and motility, and led to cell death. DA appeared to have a multi-targeted approach to cellular structures and multiple life processes, leading to cell death. The results of this study could provide molecular evidence for the analysis of the insecticidal toxicology of DA and further improve the study of the pathogenic insect mechanism of Metarhizium.

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10-4 to 10-5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects.

Destruxin A (DA) is a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus, Metarhizium anisopliae. It has insecticidal activity against host insect\'s innate immunity system, but the molecular mechanism is not yet elucidated. In our previous experiment, four HSPs (heat shock proteins, BmHSP70-3, BmHSP75, BmHSP83, and BmHSCP) were characterized from the specific protein electrophoretic bands of Bombyx mori Bm12 cell line treated with DA in the test of drug affinity responsive target stability (DARTS), which implied that these HSPs might be kinds of DA-affinity proteins, or DA induces them up-regulated expression. Therefore, in current research, the interactions of DA and HSPs were explored through analysis of bio-layer interferometry (BLI) employing FortBio OcteteQK. The expression levels of HSPs genes were surveyed by quantitative real-time polymerase chain reaction (qPCR). The results indicated that DA had no interactions with BmHSP70-3, BmHSP75, and BmHSP83, but had affinity to BmHSCP with a KD value of 88.1 μM, in BLI analysis. However, the expression levels of all HSPs genes were significantly up-regulated after the Bm12 cells were treated by DA. In conclusion, DA can induce the four HSPs expression in Bm12 cells, but DA only binds to BmHSCP. Our research provides new insights on understanding of the action mechanisms of destruxins.

Destruxin A (DA), a cyclodepsipeptidic mycotoxin of entomopathogenic fungus, Metarhizium anisopliae, has anti-immunity activity against insects, but the mechanism of immune regulation is not clear yet. In our previous experiment, the significant expression changes of Bm_nscaf2838_045, Bm_nscaf2674_066, and Bm_nscaf2767_133 genes in a silkworm\'s hemocytes were found, which suggested that these genes might be involved in insect\'s innate immunity. In the current experiment, the silkworm cell line Bm12 was used to survey the expression levels of these genes after the cells were treated with DA and the transcription factors BmRel, BmRelish1 and BmRelish2 were silenced by specific siRNA. The results indicated that, after the cells were treated by DA, the gene expression level of BmRelish2 was significantly downregulated, but BmRel and BmRelish1 were not changed. The results also showed that the gene expression levels of Bm_nscaf2838_045 and Bm_nscaf2674_066 had similar phenomena, i.e., downregulation with individual BmRelish1 gene silence or DA treatment, upregulation with combination of BmRelish1 gene silence and DA treatment, upregulation with individual BmRelish2 gene silence, and downregulation with combination of BmRelish2 gene silence plus DA treatment, but no changes in the BmRel gene silence combined with DA treatment. For the Bm_nscaf2767_133 gene, the downregulated expressions were found in individual BmRelish2 gene silence or DA treatment, upregulation in the combination treatment of BmRelish2 gene silence plus DA, and the individual treatment of BmRel or BmRelish1 silence. It is suggested that expressions of the Bm_nscaf2838_045 and Bm_nscaf2674_066 genes are closely related to the Imd signal pathway, but Bm_nscaf2767_133 genes might involve in both Toll and Imd pathways. Furthermore, the BmRelish1 gene acts as an activator and the BmRelish2 gene acts as a repressor for both Bm_nscaf2838_045 and Bm_nscaf2674_066 gene expressions. It also implies that DA may participate in the splicing process of BmRelish where BmRelish2 was promoted. Our research will provide new insights on the understanding of the activity mechanisms of destruxins.

The entomopathogenic fungi Metarhizium brunneum, Beauveria bassiana, and B. brongniartii are widely applied as biological pest control agent in OECD countries. Consequently, their use has to be flanked by a risk management approach, which includes the need to monitor the fate of their relevant toxic metabolites. There are still data gaps claimed by regulatory authorities pending on their identification and quantification of relevant toxins or secondary metabolites. In this chapter, analytical methods are presented allowing the qualitative and quantitative analysis of the relevant toxic B. brongniartii metabolite oosporein and the three M. brunneum relevant destruxin (dtx) derivatives dtx A, dtx B, and dtx E.

The insect pathogenic fungus Metarhizium anisopliae is an important insect biological control agent commercialized for use worldwide. Fungal infection is percutaneous, and rapid germination and growth has been linked to virulence. Using a simple in vitro growth screen to isolate mutants with increased virulence, M. anisopliae SM04 conidia were exposed to UV radiation for 20, 40, and 60 min, and mutants were subsequently screened for more rapid growth on standard potato dextrose agar. From a screen of >6,000 colonies, mutants were selected based on larger colony diameters as compared to the wild-type parent. Insect bioassays using the diamondback moth, Plutella xylostella, revealed one mutant, designated as MaUV-40.1 as displaying both more rapid growth and increased virulence. The mean lethal time to kill (LT50 using 106 conidia/ml) was 57.6 and 115.4 h for the MaUV-40.1 mutant and wild-type strains, respectively. Total conidial production, UV and thermal tolerances of the MaUV-40.1 strain were increased, but a reduced secretome was seen for the mutant compared to wild type. Analyses of culture supernatants indicated significant shifts in secondary metabolite production in the mutant. The insecticidal activity of EthOAc extracts derived from MaUV-40.1 mutant cell-free culture supernatants were ~20 times more potent that wild-type extracts. These data indicate that mutagenesis coupled to a growth screen can be a simple approach to isolate strains with greater stress resistance and virulence and that cell-free extracts may hold promise as an alternative to the living organism for insect control.

Destruxins, cyclic hexadepsipeptide toxins, secreted by the entomopathogenic fungus, Metarhizium anisopliae through extracellular synthesis. The present study reports a new approach for the analysis of DTXs produced by the fungal strain Metarhizium anisoliae Tk6, using FRIR-HPLC-LC-MS and H(1) NMR. The results also showed that production of the major DTXs A, B, C, and E have to be determined in Czapek Dextrose (CD) liquid culture filtrate from 9 to 12 days post-inoculation. Purified DTX were further tested in bioassays to assess their effects of Aedes aegypti mosquitoes. The four major purified DTX compounds were found to cause a toxic effect on the larval developmental stages of mosquitoes with high mortality rates. However, DTX E outperformed the other three DTXs by causing the highest mortality three days after inoculation. This result gives an alternative approach of using DTXs in mosquitoes control and used as a new method for other pest management.

The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions. Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non-sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes. Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly. We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier.
Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.