Metarhizium brunneum infection dynamics differ at the cuticle interface of susceptible and tolerant morphs of Galleria mellonella

ABSTRACT

In order for entomopathogenic fungi to colonize an insect host, they must first attach to, and penetrate, the cuticle layers of the integument. Herein, we explored the interactions between the fungal pathogen Metarhizium brunneum ARSEF 4556 and two immunologically distinct morphs, melanic (M) and non-melanic (NM), of the greater wax moth Galleria mellonella. We first interrogated the cuticular compositions of both insect morphs to reveal substantial differences in their physiochemical properties. Enhanced melanin accumulation, fewer hydrocarbons, and higher _L-dihydroxyphenylalanine (DOPA) decarboxylase activity were evident in the cuticle of the M larvae. This “hostile” terrain proved challenging for M. brunneum – reflected in poor conidial attachment and germination, and elevated expression of stress-associated genes (e.g., Hsp30, Hsp70). Lack of adherence to the cuticle impacted negatively on the speed of kill and overall host mortality; a dose of 10⁷ conidia killed ~30% of M larvae over a 12-day period, whereas a 100-fold lower dose (10⁵ conidia) achieved a similar result for NM larvae. Candidate gene expression patterns between the insect morphs indicated that M larvae are primed to “switch-on” immunity-associated genes (e.g., phenoloxidase) within 6–12 h of conidia exposure and can sustain a “defense” response. Critically, M. brunneum responds to the distinct physiochemical cues of both hosts and adjusts the expression of pathogenicity-related genes accordingly (e.g., Pr2, Mad1, Mad2). We reveal previously uncharacterized mechanisms of attack and defence in fungal-insect antibiosis.

KEYWORDS:

• Innate immunity
• host-pathogen interactions
• cuticle
• entomopathogenic fungi
• melanization
• Galleria mellonella

Acknowledgments

We thank Dr Christopher Cunningham (Swansea University) for access to the (unpublished) preliminary annotation of the G. mellonella genome. We thank Dr. Olga Polenogova and Dr. Vadim Kryukov for help with pilot experiments of wax moth infection, and Carolyn Greig (Swansea University) for some technical support.

Author contributions

CC, ID, and TB conceived and designed the experiments, and provided resources. EG and ID performed the experiments. EG, ID, and CC collated and analysed the data. CC, ID, and TB wrote the manuscript. CC revised the manuscript.

Datasets are available on request

The raw data supporting the conclusions of this manuscript will be made available by the authors (ID and EG), without undue reservation, to any qualified researcher.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplemental material

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Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

TMB secured grant funding from the Biotechnology and Biological Sciences Research Council, the Department for Environment, Food and Rural Affairs, the Economic and Social Research Council, the Forestry Commission, the Natural Environment Research Council and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. ID and EG obtained funding from the RFBR (research projects №18-316-20007 and №19-016-00121) and the Russian Science Foundation (project no. 19-16-00019 for qPCR work). CJC’s financial contributions were facilitated by start-up funds from the College of Science, Swansea University.