Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants’ fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.


Sampling for middens
We also sampled the middens produced by queenright colonies and queenless colonies to observe if the ants were removing these fungi from their fungus gardens. We plated five fragments of midden from each queenright colony and queenless colony on Petri dishes containing PDA 20% and then incubated these at 25°C for 15 days. This procedure was conducted 24 hours after inoculation of conidial suspensions for all treatments (Day 1) and then every 72 hours, totalling 10 days of sampling. We checked the plates daily to verify the occurrence of Escovopsis, Escovopsioides and Trichoderma.
In the same manner as the previous variable (sampling of fungus gardens), we recognized that the ideal would be to identify these fungal genera to species level; this was not possible, however, for practical reasons. No growth of Escovopsis and Escovopsioides was observed in the midden of queenright or queenless colonies. We suspect that midden could present these fungi, nevertheless, other microorganisms that are also present may have inhibited their growth in the culture medium. On the other hand, Trichoderma was found growing the midden from the queenright colonies and queenless colonies, independent of the treatment to which they were exposed.
Supplementary Figure 1. Frequency of the fungal genera Escovopsis, Escovopsioides and Trichoderma found in the midden of (A) queenright colonies and (B) queenless colonies of Acromyrmex subterraneus subterraneus leafcutter ants exposed to one of three treatments or to the blank control: conidial suspension of the fungi Escovopsis moelleri (E); Escovopsioides nivea (O); Trichoderma longibrachiatum (T); blank control (0.01% Tween 80® solution + saline solution -NaCl 0.85%; C). The bars represent the mean frequency of presence of these fungi in the 10 days of sampling. Last day of weighing and sampling of queenright colonies and queenless colonies; Last day of weighing the leaves cut by the ants from queenright colonies and queenless colonies; Day 118 End of the experiment: last day of survival evaluation.
Reaction results of the ITS region were 23 sequenced in ABI3500 (Life Technologies) and LSU and tef1 regions were sent to Macrogen Inc (dna.macrogen.com/eng/, Seoul, S. Korea) for purification and sequencing. Sequences were attached in contigs with BioEdit v. 7.1.3 (Hall, 1999).
For Trichoderma molecular identification, we prepared monosporic cultures from the isolate VIMI 17.0135. The isolate was grown in PDA 20% medium for 7 days in the dark. The mycelium was collected from the agar surface and macerated in liquid nitrogen. Genomic DNA from the sample was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, USA), following the manufacturer's protocol. DNA quality was then analysed with agarose gel electrophoresis (1%) and quantified with Nanodrop 1000ND (Thermo Scientific, USA). We amplified the ITS region, using the primers ITS 1 (5'-TCCGTAGGTGAACCTGCGG -3') and ITS 4 (5'-TCCTCCGCTTATTGATATGC -3') (White et al., 1990).