Cordycepin and the entomopathogenic fungus Cordyceps militaris

Cordyceps militaris is a widespread entomopathogenic fungus found in Europe, Asia, and North America, with a large number of insect hosts, predominantly lepidopteran larvae (caterpillars). This species is well known for its production of the nucleoside analogue cordycepin (3’-deoxyadenosine). Co-produced with its protector molecule pentostatin, cordycepin is a polyadenylation inhibitor, via its active modified form as cordycepin triphosphate. Pentostatin protects cordycepin from degradation to 3’-deoxyinosine by inhibition of the enzyme adenosine deaminase. It has been shown to have anti-inflammatory effects and hence has been the subject of much pharmacological research. Until recently, very little was known about the role of cordycepin in the ecology of the fungus, or why its production is favoured by natural selection. There are also gaps in the understanding of the process of infection of the host by the fungus, which is directly followed by sexual development and the formation of stromata bearing sexual fruiting bodies and spores (ascospores). Understanding these areas could have implications for biological control of insect pests. Indeed, the related species Beauveria bassiana and Metarhizium anisopliae have been used as bioinsecticides, precluding the use of harmful chemical insecticides. Culture degeneration is a phenomenon defined previously as a reduction in the production of cordycepin by C. militaris. Experiments comparing a degenerated strain of an isolate of C. militaris with its parental control strain were performed, involving the use of gene expression analysis and metabolomics. Reduced cordycepin production in the degenerated strain was shown to be accompanied by declines in sexual development-related gene expression, and reduced production of other metabolites involved in the citrate cycle and purine metabolism. This suggested a link between cordycepin production, primary metabolism, and sexual development. We hypothesised that the production of cordycepin by C. militaris aids the infection of the insect by suppression of the host immune system, and that pentostatin, by providing molecular protection, enhances this effect. In a caterpillar infection assay system involving the injection of spores into the model species Galleria mellonella (greater wax moth) caterpillars, the lower cordycepin-producing degenerated strain was shown to produce a significantly-decreased pathogenic response, marked by reduced fungal growth in the host. When spores of the degenerated strain were supplemented by cordycepin and pentostatin, fungal emergence rates and levels significantly increased, restoring the infection performance of the degenerated strain to that of the parental control. Assays of insect gene expression were also performed, and cordycepin was demonstrated to suppress the upregulation of immune response genes in both Drosophila melanogaster Schneider 2 cells and G. mellonella haemolymph cells. Pentostatin enhanced the effects of low cordycepin concentrations in both models. These findings support the hypothesis that cordycepin has an important role in aiding insect infection by the fungus via immune suppression, and that the effect of cordycepin on host cell responses is maintained by pentostatin. Biosynthesis genes (Cns genes) for cordycepin and pentostatin are located in the same gene cluster. We hypothesised that cordycepin-pentostatin co-production was a rare trait, and its evolution had been resultant partly due to horizontal gene transfer between different species. This was due to the lack of cordycepin in other Cordyceps species, and genetic evidence of its production only found previously in two other, distantly-related species. Bioinformatics work involving tBLASTn searches through the sequenced genomes of over two and a half thousand fungal species uncovered evidence of homologous Cns gene clusters in five new species. This together with consideration of protein structures suggests that the development of cordycepin-pentostatin co-production has occurred by convergent evolution involving duplication and subfunctionalisation of genes involved in the purine synthesis pathway, and/or through horizontal gene transfer

Culture degeneration reduces sex-related gene expression, alters metabolite production and reduces insect pathogenic response in Cordyceps militaris

Cordyceps militaris is an entomopathogenic ascomycete, known primarily for infecting lepidopteran larval (caterpillars) and pupal hosts. Cordycepin, a secondary metabolite produced by this fungus has anti-inflammatory properties and other pharmacological activities. However, little is known about the biological role of this adenosine derivate and its stabilising compound pentostatin in the context of insect infection the life cycle of C. militaris. During repeated subcultivation under laboratory conditions a degeneration of C. militaris marked by decreasing levels of cordycepin production can occur. Here, using degenerated and parental control strains of an isolate of C. militaris, we found that lower cordycepin production coincides with the decline in the production of various other metabolites as well as the reduced expression of genes related to sexual development. Additionally, infection of Galleria mellonella (greater wax moth) caterpillars indicated that cordycepin inhibits the immune response in host haemocytes. Accordingly, the pathogenic response to the degenerated strain was reduced. These data indicate that there are simultaneous changes in sexual reproduction, secondary metabolite production, insect immunity and infection by C. militaris. This study may have implications for biological control of insect crop pests by fungi

Cordycepin and the entomopathogenic fungus Cordyceps militaris

Cordyceps militaris is a widespread entomopathogenic fungus found in Europe, Asia, and North America, with a large number of insect hosts, predominantly lepidopteran larvae (caterpillars). This species is well known for its production of the nucleoside analogue cordycepin (3’-deoxyadenosine). Co-produced with its protector molecule pentostatin, cordycepin is a polyadenylation inhibitor, via its active modified form as cordycepin triphosphate. Pentostatin protects cordycepin from degradation to 3’-deoxyinosine by inhibition of the enzyme adenosine deaminase. It has been shown to have anti-inflammatory effects and hence has been the subject of much pharmacological research. Until recently, very little was known about the role of cordycepin in the ecology of the fungus, or why its production is favoured by natural selection. There are also gaps in the understanding of the process of infection of the host by the fungus, which is directly followed by sexual development and the formation of stromata bearing sexual fruiting bodies and spores (ascospores). Understanding these areas could have implications for biological control of insect pests. Indeed, the related species Beauveria bassiana and Metarhizium anisopliae have been used as bioinsecticides, precluding the use of harmful chemical insecticides. Culture degeneration is a phenomenon defined previously as a reduction in the production of cordycepin by C. militaris. Experiments comparing a degenerated strain of an isolate of C. militaris with its parental control strain were performed, involving the use of gene expression analysis and metabolomics. Reduced cordycepin production in the degenerated strain was shown to be accompanied by declines in sexual development-related gene expression, and reduced production of other metabolites involved in the citrate cycle and purine metabolism. This suggested a link between cordycepin production, primary metabolism, and sexual development. We hypothesised that the production of cordycepin by C. militaris aids the infection of the insect by suppression of the host immune system, and that pentostatin, by providing molecular protection, enhances this effect. In a caterpillar infection assay system involving the injection of spores into the model species Galleria mellonella (greater wax moth) caterpillars, the lower cordycepin-producing degenerated strain was shown to produce a significantly-decreased pathogenic response, marked by reduced fungal growth in the host. When spores of the degenerated strain were supplemented by cordycepin and pentostatin, fungal emergence rates and levels significantly increased, restoring the infection performance of the degenerated strain to that of the parental control. Assays of insect gene expression were also performed, and cordycepin was demonstrated to suppress the upregulation of immune response genes in both Drosophila melanogaster Schneider 2 cells and G. mellonella haemolymph cells. Pentostatin enhanced the effects of low cordycepin concentrations in both models. These findings support the hypothesis that cordycepin has an important role in aiding insect infection by the fungus via immune suppression, and that the effect of cordycepin on host cell responses is maintained by pentostatin. Biosynthesis genes (Cns genes) for cordycepin and pentostatin are located in the same gene cluster. We hypothesised that cordycepin-pentostatin co-production was a rare trait, and its evolution had been resultant partly due to horizontal gene transfer between different species. This was due to the lack of cordycepin in other Cordyceps species, and genetic evidence of its production only found previously in two other, distantly-related species. Bioinformatics work involving tBLASTn searches through the sequenced genomes of over two and a half thousand fungal species uncovered evidence of homologous Cns gene clusters in five new species. This together with consideration of protein structures suggests that the development of cordycepin-pentostatin co-production has occurred by convergent evolution involving duplication and subfunctionalisation of genes involved in the purine synthesis pathway, and/or through horizontal gene transfer

Data from: Consequences of symbiont co-infections for insect host phenotypes

1. Most animals host communities of symbiotic bacteria. In insects, these symbionts may have particularly intimate interactions with their hosts: many are intracellular and can play important roles in host ecology and evolution, including protection against natural enemies. 2. We investigated how interactions between different species or strains of endosymbiotic bacteria within an aphid host influence the outcome of symbiosis for both symbiont and host. 3. We first asked whether different combinations of facultative symbiont species or strains can exist in stable co-infections. We then investigated whether the benefits that facultative bacteria confer on their hosts (protection against natural enemies) are enhanced, reduced or unaltered by the presence of a co-infecting symbiont. We asked this both for co-infecting symbionts that confer different phenotypes on their hosts (protection against fungal pathogens vs. parasitoid wasps) and symbionts with overlapping functions. Finally, we investigated the additional survival costs to aphids of carrying multiple infections of symbiont species or strains, and compared symbiont titres in double and single infections. 4. We found that stable co-infections were possible between all of the combinations of facultative symbiont species (Regiella insecticola + Hamiltonella defensa, Regiella + Rickettsiella sp., Regiella + Spiroplasma sp.) and strains (Hamiltonella) that we studied. Where symbionts provided protection against different natural enemies, no alteration in protection was observed in the presence of co-infections. Where symbionts provided protection against the same natural enemy, the level of protection corresponded to the higher of the two symbionts present. In some instances, aphid hosts suffered additional survival costs when hosting double infections. In the case of Hamiltonella, however, infection with multiple strains of the same symbiont led to lower symbiont titres than single infections, and actually improved aphid survival. 5. We conclude that the long-term maintenance of symbiont co-infections in aphids is likely to be determined primarily by costs of co-infections and in some instances by redundancy of symbiont benefits