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.

Background Circadian clocks allow organisms to anticipate daily fluctuations in their environment by driving rhythms in physiology and behavior. Inter-organismal differences in daily rhythms, called chronotypes, exist and can shift with age. In ants, age, caste-related behavior and chronotype appear to be linked. Brood-tending nurse ants are usually younger individuals and show “around-the-clock” activity. With age or in the absence of brood, nurses transition into foraging ants that show daily rhythms in activity. Ants can adaptively shift between these behavioral castes and caste-associated chronotypes depending on social context. We investigated how changes in daily gene expression could be contributing to such behavioral plasticity in Camponotus floridanus carpenter ants by combining time-course behavioral assays and RNA-Sequencing of forager and nurse brains. Results We found that nurse brains have three times fewer 24 h oscillating genes than foragers. However, several hundred genes that oscillated every 24 h in forager brains showed robust 8 h oscillations in nurses, including the core clock genes Period and Shaggy. These differentially rhythmic genes consisted of several components of the circadian entrainment and output pathway, including genes said to be involved in regulating insect locomotory behavior. We also found that Vitellogenin, known to regulate division of labor in social insects, showed robust 24 h oscillations in nurse brains but not in foragers. Finally, we found significant overlap between genes differentially expressed between the two ant castes and genes that show ultradian rhythms in daily expression. Conclusion This study provides a first look at the chronobiological differences in gene expression between forager and nurse ant brains. This endeavor allowed us to identify a putative molecular mechanism underlying plastic timekeeping: several components of the ant circadian clock and its output can seemingly oscillate at different harmonics of the circadian rhythm. We propose that such chronobiological plasticity has evolved to allow for distinct regulatory networks that underlie behavioral castes, while supporting swift caste transitions in response to colony demands. Behavioral division of labor is common among social insects. The links between chronobiological and behavioral plasticity that we found in C. floridanus, thus, likely represent a more general phenomenon that warrants further investigation.

s Ophiocordyceps fungi manipulate ant behaviour as a transmission strategy. Conspicuous changes in the daily timing of disease phenotypes suggest that Ophiocordyceps and other manipulators could be hijacking the host clock. We discuss the available data that support the notion that Ophiocordyceps fungi could be hijacking ant host clocks and consider how altering daily behavioural rhythms could benefit the fungal infection cycle. By reviewing time‐course transcriptomics data for the parasite and the host, we argue that Ophiocordyceps has a light‐entrainable clock that might drive daily expression of candidate manipulation genes. Moreover, ant rhythms are seemingly highly plastic and involved in behavioural division of labour, which could make them susceptible to parasite hijacking. To provisionally test whether the expression of ant behavioural plasticity and rhythmicity genes could be affected by fungal manipulation, we performed a gene co‐expression network analysis on ant time‐course data and linked it to available behavioural manipulation data. We found that behavioural plasticity genes reside in the same modules as those affected during fungal manipulation. These modules showed significant connectivity with rhythmic gene modules, suggesting that Ophiocordyceps could be indirectly affecting the expression of those genes as well.

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.

Background: Behavioral plasticity in the nocturnal ant Camponotus floridanus is associated with changes in daily rhythms of core clock and clock-controlled genes in the brain. Plasticity in clock-controlled output, although adaptive, has been hypothesized to be a target for parasites that change host behavior in a timely manner to complete their life cycle. This study aims to explore this hypothesis by characterizing how the transcriptomic rhythms of the ant host change upon infection by a behavior manipulating parasite. We compared and contrasted the daily gene expression profile of uninfected C. floridanus ant heads to ants infected by a manipulating fungal parasite Ophiocordyceps camponoti-floridani and a non-manipulating fungus Beauveria bassiana, to test if changes to host clock and clock-controlled gene expression are specific to behavioral modifying diseases, or if such changes are a general hallmark of infectious diseases. Results: The repertoire of genes oscillating every 24h in the ant heads showed almost three-fold reduction during O. camponoti-floridani infections, as compared to uninfected controls. Control-like nocturnal activity of 24h-rhythmic genes was maintained during O. camponoti-floridani infections, but not in B. bassiana infected ant heads. Half of all genes that showed 24h rhythms in the heads and brains of uninfected ants displayed highly synchronized changes in their rhythmic expression during both diseases, but in a species-specific manner. Network analyses revealed that both fungal parasites affected the same links between behavioral plasticity and clock output, albeit in a different manner. Conclusion: Changes to clock-controlled transcriptomic rhythms of hosts might be a general hallmark of infectious diseases. However, the infection-associated changes to clock-controlled rhythms of the host are species-specific, and likely depends on the life history strategies used by the parasite.