Pre-biting behavior
The ant species that is the primary host of Ophiocordyceps unilateralis s.l. at our field site is Camponotus leonardi[23]. This ant is canopy dwelling, rarely descending to the forest floor and when it does it always travels on well defined trails (Additional File 1). Trail individuals do not forage on the forest floor and trails normally ascend into the canopy within 3-5 m from where they descended (suggesting that workers descend only because breaks in the canopy necessitate a descent to reach adjacent foraging crowns in the canopy). Unlike ants on trails the manipulated ants in the pre-leaf biting stage were all discovered walking alone on low vegetation, usually on saplings <50 cm above soil level and only during the time interval 09:30-12:45 h (n = 21, Figure 1 and 2). All 21 zombie ants that we followed were confirmed to be infected either via dissection of the head to reveal fungal cells or by observing the emergence of O. unilateralis s.l. following death on the leaf (Figure 3a). Post mortem fungal growth starts with abundant hyphae emanating from the intersegmental membranes within 2-3 days after host death and ultimately leads to stalk formation from the back of the ant's head [20].
The host ant is diurnal at our field site [23] and infected ants (n = 42) appeared even more restricted in their activity as they were never observed in the early morning or late afternoon (15:00-18:00 hrs), in spite of our searches covering these early and late periods of the day. The understory vegetation of our study site was extensively searched during a year-long census program that examined every leaf below 2 m height in 1360 m2 of forest habitat [23]. We therefore conclude that pre-biting infected C. leonardi ants at this site were only active in the morning and that this observation was not affected by sampling bias. The occasional trails of healthy ants that can be found on the forest floor (Additional File 1) were observed both during the morning hours and in the late afternoon, with activity on trails always ceasing around sunset, i.e. between 17:00-18:00 h.
Since behavioral manipulation alters normal behavior we could not a priori exclude the possibility that infected individuals would have become nocturnal. We therefore conducted evening (after 18:00 h) and night (22:00-0:00 h) surveys using torches, but did not find any C. leonardi ants active in the dark. Furthermore, a collected colony of C. leonardi that likely contained some naturally infected ants was maintained under field laboratory conditions for 2 days and did not show any activity in the dark, suggesting that behaviorally manipulated C. leonardi ants remain only active during daylight.
We also performed 20 hours and 28 minutes of focal observations on 12 infected ants that were found walking alone (infection status was later confirmed as described above)(see Figure 1, individuals 10-21). These individuals all expressed irregularly spaced whole body convulsions (vertical bars on the periodogram in Figure 1), which often made the ant fall from the vegetation onto the ground (denoted as stars on the periodogram). After falling infected ants always resumed walking and always climbed a small sapling or comparable plant, which were abundantly present in the understory.
We never observed trail ants falling from vegetation. To document this, we removed 13 such ants from a trail on a liana approximately 1 m above the forest floor in the same area where we observed the behaviorally manipulated ants. The liana descended from the canopy and the trail ascended into the canopy via a tree trunk less than 3 m from where we collected the ants. The trail ants (assumed to be uninfected) were placed on the ground and they all quickly ascended into the canopy where we collected them again from tree trunks ca. 1.5 m above ground. The only exception was one trail ant that was predated upon by a spider (none of the behaviorally manipulated ants we observed were predated upon). Trail ants did not spend extensive time walking in the understory. Their median time between release and reaching the trunk on which they ascended into the canopy was 28 minutes (range 7-51, total observation time 6 h, 2 m). After these observations, the collected trail ants were maintained singly without food and died within a few days without signs of O. unilateralis s.l. fungal growth.
Before biting a leaf, infected ants were predominantly walking (average proportion of time walking 0.62, range 0.11-1.00, total observation time 15 h, 35 m). They traversed an average of 99 leaves (range 52-239, 8 focal ants), which was ca. twice the number traversed by trail ants (average 51, range 8-140, 13 focal ants). Because trail ants were never observed walking on leaves except at the times when we removed them from trails and placed them on the ground we conclude that traversing leaves is not a normal behavior. Therefore we did not statistically test for a difference between the numbers of leaves traversed, as this was not biologically meaningful.
During the pre-biting phase behaviorally manipulated ants appeared to express a random "drunkard's walk" such that an individual remained close to its starting point [29] but precise trajectories were not mapped so this remains a heuristic assessment. In all cases the infected ants finally bit into leaves <3 m from where they were first observed.
The timing at which infected ants bit into leaves was synchronised around noon (Figure 1 & 2; n = 16), suggesting either a direct solar cue or an indirect one via correlated temperature or humidity. The solar elevation at the moment of biting was 80.28° +/- 1.32 SE, which was close to the maximum solar elevation of 87.29° +/- 0.39 SE during our study period. Once they had bitten leaves, ants rarely became detached and when this happened it was due to disturbance (two cases, # 15 and 18 in Figure 1, after very heavy rain). Biting leaves is not part of the repertoire of healthy ants of this species.
Post-death grip behavior
After biting into leaves infected ants always died as this is a developmental necessity for the subsequent growth of the fungus [15, 20]. It was not possible to determine the exact time of death since obvious signs such as muscle activity could be the result of fungal action, but it did appear that ants could remain alive for as long as six hours after biting. Video recordings of six live ants biting leaves revealed very little behavior of interest besides a periodic twitching of the legs (Additional File 2). The arrival of an ant of a different species close to a biting ant provoked no responses (Additional File 2), in contrast to healthy ants on trails, which were very aggressive to other ant species they encountered at food sources as well as to flying insects like wasps and flies that landed near honey baits.
Muscular atrophy accompanies behavioral manipulation
At the moment of the death grip, when the ant is under fungal control and biting into the major vein of a leaf its head is filled with fungal cells (Figure 3). These cells, called hyphal bodies, were very abundant and could be found between the muscle fibers and surrounding the brain and post pharyngeal gland (Figure 3), but not inside muscles, brains or glands.
The most prominent other sign of infection, besides the abundance of fungal cells inside the head capsule, was that the mandibular muscles were atrophied. We sectioned the heads of 10 ants that were biting leaves and the pathology was the same across all 10. Mandibular muscle fibers, which normally attach to the head capsule, often appeared to have become detached (Figure 3c) and where fibers remained attached they were stretched (compare 3b and 3c). Ant workers have both mandibular opening and closing muscles and these can be discriminated in healthy ants by their typical length of sarcomeres: 2-3 μm for opening muscles and 5-6 μm for closing muscles (Figure 3b). However, in parasitized ants the characteristic stretching of sarcomeres made it impossible to accurately distinguish between these two types of muscles. This may imply that fungal effects on these muscles are unlikely to be cell specific at the time of biting. Our behavioral observations revealed that the mandibles worked normally in the hours preceding the death grip as infected ants were observed to self groom, cleaning their antennae and legs, which involves precise opening and closing of the mandibles as these appendages are pulled across the maxillae to be cleaned.
At the sub-cellular level (as seen with TEM) the muscles of infected ants were very distinct from those of healthy individuals (Figure 4). Striated muscles (such as the mandible muscles) are composed of fibers that are multinucleated cells formed as a result of cell fusion. These fibers contain thick (myosin) and thin (actin) filaments which attach during cross-bridge cycling leading to muscle shortening. To achieve contraction mitochondria and sarcoplasmic reticulum provide energy (ATP) and ionic calcium (Ca++), respectively. At the end of each sarcomere unit there is a z-line (sarcomeres are in fact defined as the area between z-lines), which can be thought of as the anchor points for muscle contraction. Infected ants sampled during the death grip had broken z-lines and significantly less dense sarcoplasmic reticulum and mitochondria. This was determined from a measurement of the increase of interfibrillar spaces that appears following the loss of organelles, which in this case are sarcoplasmic reticulum and mitochondria (Kruskal-Wallis test, 20.25, df = 1, p < 0.0001, n = 6, Figure 4e). Similar to the light micrographs, the transmission electron micrographs showed a distinct atrophy in the muscles of infected ants.
Despite the apparent atrophy of muscles the behaviorally manipulated ants were able to exert considerable force. We removed 29 dead ants from diverse species of monocotyledonous and dicotyledonous leaves collected at our site (n = 10 and 19, respectively). On each leaf large puncture wounds were evident where the ant mandible had penetrated into the leaf (Figure 5). Only in two cases was the major vein on which biting was centered not cut into.