Project description:The entomopathogenic fungus Metarhizium acridum in oil-based formulations (Green Muscle® (GM)) is a biopesticide for locust control lacking side-effects on biodiversity, unlike chemical insecticides. Under controlled conditions, GM-treated locusts and grasshoppers attract predators, a complementary advantage in locust control. We assessed avian predation on a population of desert locusts in northern Niger aerially sprayed operationally with GM with 107 g viable conidia ha-1. Populations of adult locusts and birds and vegetation greenness were assessed simultaneously along two transects from 12 days before until 23 days after treatment. Common kestrels Falco tinnunculus and lanners F. biarmicus were the predominant avian predators. Regurgitated pellets and prey remains were collected daily beneath "plucking posts" of kestrels. Locusts started dying five days post-spray and GM had its maximum effect one-two weeks after the spray, with 80% efficacy at day 21. After spraying, bird numbers increased significantly (P<0.05) concurrent with decreasing desert locust densities. Locust numbers decreased significantly (P<0.001) with both time since spraying and decreasing greenness. Before spraying, kestrel food remains under plucking posts accounted for 34.3 ±13.4 prey items day-1, of which 31.0 ±11.9 were adult desert locusts (90.3%), reducing post-spray to 21.1 ±7.3 prey items day-1, of which19.5 ±6.7 were adult desert locusts (92.5%), attributable to decreased use of the plucking-posts by the kestrels rather than an effect of the spray. After spraying, kestrels took significantly (P<0.05) more larger female (75-80%) than smaller male (20-25%) locusts. Avian predation probably enhanced the impact of the GM on the desert locust population, especially by removing large adult females. No direct or indirect adverse side-effects were observed on non-target organisms including locust predators such as ants and birds. These substantial ecological advantages should also be considered when choosing between conventional chemical and biopesticide-based locust control.
Project description:BACKGROUND: The entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl) hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering. RESULTS: We selected four Ntl over-expression and four Ntl RNA interference (RNAi) transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type. CONCLUSIONS: Ntl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence.
Project description:Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A and B, which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A showed cytotoxicity to three cancer lines with IC??'s of 6.2, 11.0, and 10.8 ?M against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B had an IC?? of 18.2 ?M against HepG2/C3A, although it was inactive at 100 ?M against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.
Project description:BACKGROUND: The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. RESULTS: Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. CONCLUSION: MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.
Project description:An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.
Project description:Behavioural fever is a common response to immune challenge in ectotherms and confers survival benefits. However, costs accrue rapidly as body temperature rises. Thus, the magnitude of adaptive fever responses might reflect the balance of costs and benefits. We investigated behavioural fever in desert locusts, Schistocerca gregaria, infected with the entomopathogenic fungus Metarhizium acridum. We first tracked the time course of behavioural fever in infected locusts, demonstrating that body temperatures rose on the day following inoculation (day 1), and reached peak intensity on the day after that (day 2). Subsequently, the magnitude of fever responses varied during a day, and locusts tended to exhibit high-intensity fever responses in the mornings when basking was first possible. We speculate that this may have resulted from increased fungal load caused by unimpeded growth overnight when locusts could not fever. We next inoculated locusts with different M. acridum doses ranging from 0 to ca. 75,000 conidia. The magnitude of their behavioural fever responses on day 2 post-inoculation was positively related to fungal dose. Thus, we demonstrate dose-dependency in the behavioural fever responses of desert locusts and suggest that this may reflect the adaptive deployment of behavioural fever to minimize costs relative to benefits.
Project description:Light is an important stimulus for fungi as it regulates many diverse and important biological processes. Metarhizium acridum is an entomopathogenic fungus currently used for the biological control of insect pests. The success of this approach is heavily dependent on tolerance to environmental stresses. It was previously reported that light exposure increases tolerance to ultraviolet radiation in M. acridum There is no information in the literature about how light globally influences gene expression in this fungus. We employed a combination of mRNA-Sequencing and high-throughput proteomics to study how light regulates gene expression both transcriptionally and post-transcriptionally. Mycelium was exposed to light for 5 min and changes at the mRNA and protein levels were followed in time-course experiments for two and four hours, respectively. After light exposure, changes in mRNA abundance were observed for as much as 1128 genes or 11.3% of the genome. However, only 57 proteins changed in abundance and at least 347 significant changes at the mRNA level were not translated to the protein level. We observed that light downregulated subunits of the eukaryotic translation initiation factor 3, the eIF5A-activating enzyme deoxyhypusine hydroxylase, and ribosomal proteins. We hypothesize that light is perceived as a stress by the cell that responds to it by reducing translational activity. Overall, our results indicate that light acts both as a signal and a stressor to M. acridum and highlight the importance of measuring protein levels in order to fully understand light responses in fungi.
Project description:As a C2H2 type zinc finger transcription factor, CreA is the key in Carbon Catabolism Repression (CCR) pathway, which negatively regulates the genes in carbon sources utilization. As conidiation in filamentous fungi is affected by nutritional conditions, CreA may contribute to fungal conidiation, which has been well studied in filamentous fungi, especially Aspergillus spp., but researches on entomopathogenic fungi are not enough. In this study, we found a homologous gene MaCreA in Metarhizium acridum, and the MaCreA deletion strain showed delayed conidiation, significant decrease in conidial yield, and 96.88% lower conidial production, when compared with the wild-type strain, and the normal conidiation and microcycle conidiation pattern shift was blocked. RT-qPCR showed that the transcription levels of the genes FlbD and LaeA (related to asexual development) were significantly altered, and those of most of the conidiation-related genes were higher in ?MaCreA strain. The results of RNA-Seq revealed that MaCreA regulated the two conidiation patterns by mediating genes related to cell cycle, cell division, cell wall, and cell polarity. In conclusion, CreA, as a core regulatory gene in conidiation, provides new insight into the mechanism of conidiation in entomopathogenic fungi.