Three-dimensional visualization and a deep-learning model reveal complex fungal parasite networks in behaviorally manipulated ants.

Some microbes possess the ability to adaptively manipulate host behavior. To better understand how such microbial parasites control animal behavior, we examine the cell-level interactions between the species-specific fungal parasite Ophiocordyceps unilateralis sensu lato and its carpenter ant host (Camponotus castaneus) at a crucial moment in the parasite's lifecycle: when the manipulated host fixes itself permanently to a substrate by its mandibles. The fungus is known to secrete tissue-specific metabolites and cause changes in host gene expression as well as atrophy in the mandible muscles of its ant host, but it is unknown how the fungus coordinates these effects to manipulate its host's behavior. In this study, we combine techniques in serial block-face scanning-electron microscopy and deep-learning-based image segmentation algorithms to visualize the distribution, abundance, and interactions of this fungus inside the body of its manipulated host. Fungal cells were found throughout the host body but not in the brain, implying that behavioral control of the animal body by this microbe occurs peripherally. Additionally, fungal cells invaded host muscle fibers and joined together to form networks that encircled the muscles. These networks may represent a collective foraging behavior of this parasite, which may in turn facilitate host manipulation.

Zombie ant death grip due to hypercontracted mandibular muscles.

There are numerous examples of parasites that manipulate the behavior of the hosts that they infect. One such host-pathogen relationship occurs between the 'zombie-ant fungus' Ophiocordyceps unilateralis sensu lato and its carpenter ant host. Infected ants climb to elevated locations and bite onto vegetation where they remain permanently affixed well after death. The mandibular muscles, but not the brain, of infected ants are extensively colonized by the fungus. We sought to investigate the mechanisms by which O. unilateralis s.l. may be able to influence mandibular muscle contraction despite widespread muscle damage. We found that infected muscles show evidence of hypercontraction. Despite the extensive colonization, both motor neurons and neuromuscular junctions appear to be maintained. Infection results in sarcolemmal damage, but this is not specific to the death grip. We found evidence of precise penetration of muscles by fungal structures and the presence of extracellular vesicle-like particles, both of which may contribute to mandibular hypercontraction.

Gene expression during zombie ant biting behavior reflects the complexity underlying fungal parasitic behavioral manipulation.

BACKGROUND: Adaptive manipulation of animal behavior by parasites functions to increase parasite transmission through changes in host behavior. These changes can range from slight alterations in existing behaviors of the host to the establishment of wholly novel behaviors. The biting behavior observed in Carpenter ants infected by the specialized fungus Ophiocordyceps unilateralis s.l. is an example of the latter. Though parasitic manipulation of host behavior is generally assumed to be due to the parasite's gene expression, few studies have set out to test this. RESULTS: We experimentally infected Carpenter ants to collect tissue from both parasite and host during the time period when manipulated biting behavior is experienced. Upon observation of synchronized biting, samples were collected and subjected to mixed RNA-Seq analysis. We also sequenced and annotated the O. unilateralis s.l. genome as a reference for the fungal sequencing reads. CONCLUSIONS: Our mixed transcriptomics approach, together with a comparative genomics study, shows that the majority of the fungal genes that are up-regulated during manipulated biting behavior are unique to the O. unilateralis s.l. genome. This study furthermore reveals that the fungal parasite might be regulating immune- and neuronal stress responses in the host during manipulated biting, as well as impairing its chemosensory communication and causing apoptosis. Moreover, we found genes up-regulated during manipulation that putatively encode for proteins with reported effects on behavioral outputs, proteins involved in various neuropathologies and proteins involved in the biosynthesis of secondary metabolites such as alkaloids.

Foraging ants trade off further for faster: use of natural bridges and trunk trail permanency in carpenter ants.

Trail-making ants lay pheromones on the substrate to define paths between foraging areas and the nest. Combined with the chemistry of these pheromone trails and the physics of evaporation, trail-laying and trail-following behaviours provide ant colonies with the quickest routes to food. In relatively uniform environments, such as that provided in many laboratory studies of trail-making ants, the quickest route is also often the shortest route. Here, we show that carpenter ants (Camponotus rufipes), in natural conditions, are able to make use of apparent obstacles in their environment to assist in finding the fastest routes to food. These ants make extensive use of fallen branches, twigs and lianas as bridges to build their trails. These bridges make trails significantly longer than their straight line equivalents across the forest floor, but we estimate that ants spend less than half the time to reach the same point, due to increased carriage speed across the bridges. We also found that these trails, mainly composed of bridges, are maintained for months, so they can be characterized as trunk trails. We suggest that pheromone-based foraging trail networks in field conditions are likely to be structured by a range of potentially complex factors but that even then, speed remains the most important consideration.

The metabolic alteration and apparent preservation of the zombie ant brain.

Some parasites can manipulate the behavior of their animal hosts to increase transmission. An interesting area of research is understanding how host neurobiology is manipulated by microbes to the point of displaying such aberrant behaviors. Here, we characterize the metabolic profile of the brain of an insect at the moment of the behavioral manipulation by a parasitic microbe. Our model system are ants infected with the parasitic fungus Ophiocordyceps kimflemingiae (=unilateralis), which manipulates ants to climb and bite into plant substrates, before killing the host (i.e. zombie ants). At the moment of the behavioral manipulation by the fungus, the host's brain is not invaded by the fungus which is known to extensively invade muscle tissue. We found that, despite not being invaded by the parasite, the brains of manipulated ants are notably different, showing alterations in neuromodulatory substances, signs of neurodegeneration, changes in energy use, and antioxidant compound that signal stress reactions by the host. Ergothionine, a fungal derived compound with known neuronal cytoprotection functions was found to be highly elevated in zombie ant brains suggesting the fungus, which does not invade the central nervous system, is preserving the brain.

Automated tracking and analysis of ant trajectories shows variation in forager exploration.

Determining how ant colonies optimize foraging while mitigating pathogen and predator risks provides insight into how the ants have achieved ecological success. Ants must respond to changing resource conditions, but exploration comes at a cost of higher potential exposure to threats. Fungal infected cadavers surround the main foraging trails of the carpenter ant Camponotus rufipes, offering a system to study how foragers behave given the persistent occurrence of disease threats. Studies on social insect foraging behavior typically require many hours of human labor due to the high density of individuals. To overcome this, we developed deep learning based computer vision algorithms to track foraging ants, frame-by-frame, from video footage shot under the natural conditions of a tropical forest floor at night. We found that most foragers walk in straight lines overlapping the same areas as other ants, but there is a subset of foragers with greater exploration. Consistency in walking behavior may protect most ants from infection, while foragers that explore unique portions of the trail may be more likely to encounter fungal spores implying a trade-off between resource discovery and risk avoidance.

Evidence for convergent evolution of host parasitic manipulation in response to environmental conditions.

Environmental conditions exert strong selection on animal behavior. We tested the hypothesis that the altered behavior of hosts due to parasitic manipulation is also subject to selection imposed by changes in environmental conditions over time. Our model system is ants manipulated by parasitic fungi to bite onto vegetation. We analyzed the correlation between forest type (tropical vs. temperate) and the substrate where the host bites (biting substrate: leaf vs. twigs), the time required for the fungi to reach reproductive maturity, and the phylogenetic relationship among specimens from tropical and temperate forests from different parts of the globe. We show that fungal development in temperate forests is longer than the period of time leaves are present and the ants are manipulated to bite twigs. When biting twigs, 90% of the dead ants we examined had their legs wrapped around twigs, which appears to provide better attachment to the plant. Ancestral state character reconstruction suggests that leaf biting is the ancestral trait and that twig biting is a convergent trait in temperate regions of the globe. These three lines of evidence suggest that changes in environmental conditions have shaped the manipulative behavior of the host by its parasite.

Disease in the Society: Infectious Cadavers Result in Collapse of Ant Sub-Colonies.

Despite the growing number of experimental studies on mechanisms of social immunity in ant societies, little is known about how social behavior relates to disease progression within the nests of ants. In fact, when empirically studying disease in ant societies, it is common to remove dead ants from experiments to confirm infection by the studied parasite. This unfortunately does not allow disease to progress within the nest as it may be assumed would happen under natural conditions. Therefore, the approach taken so far has resulted in a limited knowledge of diseases dynamics within the nest environment. Here we introduced a single infectious cadaver killed by the fungus Beauveria bassiana into small nests of the ant Camponotus castaneus. We then observed the natural progression of the disease by not removing the corpses of the ants that died following the first entry of the disease. Because some behaviors such as social isolation of sick individuals or the removal of cadavers by nestmates are considered social immune functions and thus adaptations at the colony level that reduce disease spread, we also experimentally confined some sub-colonies to one or two chamber nests to prevent the expression of such behaviors. Based on 51 small nests and survival studies in 1,003 ants we found that a single introduced infectious cadaver was able to transmit within the nest, and social immunity did not prevent the collapse of the small sub-colonies here tested. This was true whether ants did or did not have the option to remove the infectious cadaver. Therefore, we found no evidence that the typically studied social immunity behaviors can reduce disease spread in the conditions here tested.

Long-term disease dynamics for a specialized parasite of ant societies: a field study.

Many studies have investigated how social insects behave when a parasite is introduced into their colonies. These studies have been conducted in the laboratory, and we still have a limited understanding of the dynamics of ant-parasite interactions under natural conditions. Here we consider a specialized parasite of ant societies (Ophiocordyceps camponoti-rufipedis infecting Camponotus rufipes) within a rainforest. We first established that the parasite is unable to develop to transmission stage when introduced within the host nest. Secondly, we surveyed all colonies in the studied area and recorded 100% prevalence at the colony level (all colonies were infected). Finally, we conducted a long-term detailed census of parasite pressure, by mapping the position of infected dead ants and foraging trails (future hosts) in the immediate vicinity of the colonies over 20 months. We report new dead infected ants for all the months we conducted the census--at an average of 14.5 cadavers/month/colony. Based on the low infection rate, the absence of colony collapse or complete recovery of the colonies, we suggest that this parasite represents a chronic infection in the ant societies. We also proposed a "terminal host model of transmission" that links the age-related polyethism to the persistence of a parasitic infection.