Quantifying Metarhizium Spore Equivalents

To determine how many spore equivalents the sprayed-on ergosterol solution was equal to, we determined the ergosterol level for (1) ants that were sprayed with the ergosterol solution used in the bioassay, (2) ants exposed to on average of 10³ spores of M. robertsii R1 and (3) control-treated ants without any spores. Using the methods described below, we quantified for each treatment both the ergosterol abundance (absolute abundance in relation to the internal standard as detailed below, that is the ISTD response factor) per ant by the highly sensitive selective ion monitoring (SIM) method in the GC–MS, and the absolute spore number per exposed ant by droplet digital PCR (ddPCR). This revealed an average ISTD response factor of 0.00396 per ant exposed to 10³ spores, and a baseline value of 0.00081 for the sham-treated ants, probably reflecting natural occurrence of fungal saprophytes in the nest boxes (note that these baseline values are only detectable with the highly sensitive SIM method, and only in pools of 8 ants, as they are below detection threshold when scan mode was used, as well as in SIM-analysed individual ants). Deducting this baseline from the ergosterol abundance in the spore-exposed ants revealed an ergosterol ISTD response factor per 1,000 spores of 0.00315. Comparing this to the value of 0.95310 (the ISTD response factor of the ergosterol-sprayed ants minus the baseline) revealed that we applied the ergosterol proportion (that is, 60% of the total compound abundance per spore) of an equivalent of 3 × 10⁵ spores. Therefore, our bioassay represented a realistic infection dose for ants, for example when in contact with sporulating carcasses of their own species or their insect prey, since carcassess sporulating with Metarhizium are covered with several to even hundreds of millions (10⁶–10⁹) of spores per carcass, packed into dense spore packages, making bulk transfer of high spore numbers likely^{53 (link)–55 (link)}.