Quantification of fungal growth and destruxin A during infection of Galleria mellonella larvae by Metarhizium brunneum.

Abstract

Destruxin A is among the major secondary metabolites produced by the entomopathogenic ascomycete Metarhizium sp., and the lack of studies concerning production of destruxin A by the fungus is most likely the biggest obstacle for the registration of new fungal strains. Although several studies focus on the production of destruxin A in culture media, few studies examine destruxin A in vivo during host infection. In the current work, Galleria mellonella was used as an insect model to develop for the first time in vivo real-time PCR- and HPLC-MS-based quantification of fungal growth and metabolite production, respectively, during infection by two strains of M. brunneum. Total mortality of sixth instar G. mellonella larvae that were immersed in a suspension of 1.0×108conidiamL-1 of M. brunneum EAMa 01/58-Su or BIPESCO5 strains reached 85.5% and 78.8%, respectively, and the percentage of cadavers with fungal outgrowth was low at 12.2% and 4.4%, respectively. The average survival time of treated larvae was 5.5days for both fungal strains. Using EAMa 01/58-Su and BIPESCO5 specific primer set, real-time PCR showed that the patterns of fungal growth were different for the two strains, whereas no significant differences were detected in the number of fungal sequence copies recovered from the infected larvae. EAMa 01/58-Su and BIPESCO5 strains secreted destruxin A from days 2 to 6 and from days 2 to 5 post treatment, respectively. For EAMa 01/58-Su and BIPESCO5, the maximum titer of destruxin A in the host was on day 4 at 0.369 and 0.06µg/larva, respectively, and throughout the pathogenic process, the total production was 0.6 and 0.09µg/larva, respectively. These results demonstrated that the strains pose a low hazard, if any, to humans and the environment. The methods used in this study to quantify fungal growth and metabolite production provided valuable data to better understand the role of destruxin A during the growth of M. brunneum in the host larvae and to monitor the fate of destruxin A in food chains.