Ant-infecting Ophiocordyceps fungi are globally distributed, host manipulating, specialist parasites that drive aberrant behaviors in infected ants, at a lethal cost to the host. An apparent increase in activity and wandering behaviors precedes a final summiting and biting behavior onto vegetation, which positions the manipulated ant in a site beneficial for fungal growth and transmission. We investigated the genetic underpinnings of host manipulation by: (i) producing a high-quality hybrid assembly and annotation of the Ophiocordyceps camponoti-floridani genome, (ii) conducting laboratory infections coupled with RNAseq of O. camponoti-floridani and its host, Camponotus floridanus, and (iii) comparing these data to RNAseq data of Ophiocordyceps kimflemingiae and Camponotus castaneus as a powerful method to identify gene expression patterns that suggest shared behavioral manipulation mechanisms across Ophiocordyceps-ant species interactions. We propose differentially expressed genes tied to ant neurobiology, odor response, circadian rhythms, and foraging behavior may result by activity of putative fungal effectors such as enterotoxins, aflatrem, and mechanisms disrupting feeding behaviors in the ant.

Various parasite-host interactions that involve adaptive manipulation of host behavior display time-of-day synchronization of certain events. One example is the manipulated biting behavior observed in Carpenter ants infected with Ophiocordyceps unilateralis sensu lato. We hypothesized that biological clocks play an important role in this and other parasite-host interactions. In order to identify candidate molecular clock components, we used two general strategies: bioinformatics and transcriptional profiling. The bioinformatics approach was used to identify putative homologs of known clock genes. For transcriptional profiling, RNA-Seq was performed on 48 h time courses of Ophiocordyceps kimflemingiae (a recently named species of the O. unilateralis complex), whose genome has recently been sequenced. Fungal blastospores were entrained in liquid media under 24 h light-dark (LD) cycles and were harvested at 4 h intervals either under LD or continuous darkness. Of all O. kimflemingiae genes, 5.3% had rhythmic mRNAs under these conditions (JTK Cycle, ≤ 0.057 statistical cutoff). Our data further indicates that a significant number of transcription factors have a peaked activity during the light phase (day time). The expression levels of a significant number of secreted enzymes, proteases, toxins and small bioactive compounds peaked during the dark phase or subjective night. These findings support a model whereby this fungal parasite uses its biological clock for phase-specific activity. We further suggest that this may be a general mechanism involved in parasite-host interactions.

AbstractThe ant-infecting Ophiocordyceps fungi are globally distributed, host manipulating, specialist parasites that drive aberrant behaviors in infected ants, at a lethal cost to the host. An apparent increase in activity and wandering behaviors precedes a final summiting and biting behavior on to vegetation, positioning the manipulated ant in a site beneficial for fungal growth and transmission. Notably, across Ophiocordyceps species and other known host manipulators, the molecular mechanisms underlying behavioral changes remain largely unclear. We explored possible genetic underpinnings of host manipulation by: (i) producing a hybrid assembly of the Ophiocordyceps camponoti-floridani genome, (ii) conducting laboratory infections coupled with RNAseq of both O. camponoti-floridani and its host, Campontous floridanus, and (iii) using these data for a comparative analysis to similar work performed in Ophiocordyceps kimflemingiae and Camponotus castaneus. We propose differentially expressed genes tied to ant neurobiology, odor response, circadian rhythms, and foraging behavior may be the result of putative fungal effectors such as enterotoxins, aflatrem, and mechanisms disrupting nutrition-sensing or caste-identity pathways.

1. Ophiocordyceps fungi manipulate the behaviour of their ant hosts to produce a summit disease phenotype, thereby establishing infected ant cadavers onto vegetation at elevated positions suitable for fungal growth and transmission.Multiple environmental and ecological factors have been proposed to shape the timing, positioning and outcome of these manipulations.2. We conducted a long-term field study of Ophiocordyceps camponoti-floridani infections of Camponotus floridanus ants-the Florida zombie ants. We propose and refine hypotheses on the factors that shape infection outcomes by tracking the occurrence of and fungal growth from hundreds of ant cadavers. We modelled and report these data in relation to weather, light, vegetation and attack by hyperparasites.3. We investigated environmental factors that could affect the occurrence and location of newly manipulated ant cadavers. New cadaver occurrence was preferentially biased towards epiphytic Tillandsia bromeliads, canopy openness and summer weather conditions (an interactive effect of temperature, humidity and precipitation). Furthermore, we suggest that incident light at the individual cadaver level reflects microhabitat choice by manipulated ants or selective pressure on cadaver maintenance for conditions that improve fungal survival. 4. We also asked which environmental conditions affect fungal fitness. Continued fungal development of reproductive structures and putative transmission increased with moist weather conditions (interaction of humidity and precipitation) and canopy openness, while being reduced by hyperparasitic mycoparasite infections. Moreover, under the most open canopy conditions, we found an atypical Ophiocordyceps growth morphology that could represent a plastic response to conditions influenced by high light levels.5. Taken together, we explore general trends and the effects of various ecological conditions on host and parasite disease outcomes in the Florida zombie ant system. These insights from the field can be used to inform experimental laboratoryThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Parasitic fungi are known to produce proteins that modulate virulence, alter host physiology, and trigger host responses. Due to their various effects on the host, these proteins are often considered a type of "effector" molecule, many of which act via protein-protein interactions (PPIs) between host and parasite. Cross-species PPIs between Ophiocordyceps camponoti-floridani and Camponotus floridanus likely underlie aspects of infection and parasitic behavioral manipulation in this host-parasite relationship. The fungal parasite Ophiocordyceps (zombie ant fungus) manipulates Camponotus (carpenter ant) behavior to induce a summit disease phenotype: the infected host ascends and attaches to an elevated position that promotes fungal growth and transmission. Machine learning approaches offer high-throughput methods to produce mechanistic hypotheses on how this behavioral manipulation occurs. Using D-SCRIPT to predict host-parasite PPIs, we found ca. 6,000 interactions involving 129 parasite genes that were previously found to be upregulated during manipulated summiting behavior. We analyzed the predicted PPIs to identify overrepresentation of functional annotations among these participating proteins hypothesized to mediate Ophiocordyceps-Camponotus interactions. Additionally, we compared these PPIs to predictions made by testing Camponotus proteins with secretomes from three fungi that differ from Ophiocordyceps by degrees of lifestyle and phylogeny, to find which PPIs were unique for Ophiocordyceps-ant interactions. Intriguingly, Ophiocordyceps-Camponotus specific PPIs carried strong signals for neuromodulatory G-protein coupled receptors in the host and frequent involvement of unannotated small secreted Ophiocordyceps proteins in a variety of PPIs. We also detected less specific overrepresentation of Camponotus oxidation-reduction, structural, and gene-regulatory proteins, in addition to Ophiocordyceps proteases. These hypothesized interactions include potential fungal drivers of ant infection and manipulation, whether by increasing, decreasing, or modulating host protein activity. As such, this work provides a springboard for targeted molecular investigations of the mechanisms underlying fungal manipulation of ant behavior.