僵尸蚂蚁01

RESEARCH ARTICLE Open Access Behavioral mechanisms and morphological symptoms of zombie ants dying from fungal infection David P Hughes1,2*, Sandra B Andersen2, Nigel L Hywel-Jones3, Winanda Himaman4, Johan Billen5 and Jacobus J Boomsma2 Abstract Background: Parasites that manipulate host behavior can provide prominent examples of extended phenotypes: parasite genomes controlling host behavior. Here we focus on one of the most dramatic examples of behavioral manipulation, the death grip of ants infected by Ophiocordyceps fungi. We studied the interaction between O. unilateralis s.l. and its host ant Camponotus leonardi in a Thai rainforest, where infected ants descend from their canopy nests down to understory vegetation to bite into abaxial leaf veins before dying. Host mortality is concentrated in patches (graveyards) where ants die on sapling leaves ca. 25 cm above the soil surface where conditions for parasite development are optimal. Here we address whether the sequence of ant behaviors leading to the final death grip can also be interpreted as parasite adaptations and describe some of the morphological changes inside the heads of infected workers that mediate the expression of the death grip phenotype. Results: We found that infected ants behave as zombies and display predictable stereotypical behaviors of random rather than directional walking, and of repeated convulsions that make them fall down and thus precludes returning to the canopy. Transitions from erratic wandering to death grips on a leaf vein were abrupt and synchronized around solar noon. We show that the mandibles of ants penetrate deeply into vein tissue and that this is accompanied by extensive atrophy of the mandibular muscles. This lock-jaw means the ant will remain attached to the leaf after death. We further present histological data to show that a high density of single celled stages of the parasite within the head capsule of dying ants are likely to be responsible for this muscular atrophy. Conclusions: Extended phenotypes in ants induced by fungal infections are a complex example of behavioral manipulation requiring coordinated changes of host behavior and morphology. Future work should address the genetic basis of such extended phenotypes. Keywords: extended phenotype behavioral manipulation, ants, fungi, convergent evolution, parasites Background Some parasites can adaptively take over and completely control the behavior of their hosts leading to positive fitness returns for parasite genes [1-4]. Host behavior is an extended phenotype of the parasite [5]. The degree of behavioral manipulation varies greatly across parasites from very slight alterations of pre-existing behaviors [6] to the expression of wholly novel behaviors that are never seen in healthy hosts [7]. Extended phenotypes have gained considerable prominence in community- [8], evolutionary- [9] and behavioral ecology [10]. Early studies of extended phenotypes focused on detail- ing behavioral changes and inferring whether they repre- sent adaptations for parasites or should rather be interpreted as adaptive defense mechanisms of the host or as by-products of infection [11-13]. Recently, more inte- grative approaches have emerged which includes a greater focus on the mechanisms by which behavioral changes occur. An important component is a fuller understanding of the biology of particular study systems and the timing * Correspondence: dhughes@psu.edu 1Departments of Entomology and Biology, Penn State University, PA 16802, USA Full list of author information is available at the end of the article Hughes et al. BMC Ecology 2011, 11:13 http://www.biomedcentral.com/1472-6785/11/13 © 2011 Hughes et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. of observation or experimentation, since parasite induced behavioral changes are highly dynamic [14]. Here we focus on a study system that is a dramatic example of adaptive manipulation of animal behavior by a parasite. Worker ants infected by fungal parasites belonging to the genus Ophiocordyceps express death grip behavior shortly before dying for no apparent other purpose than to assist parasite reproduction [15,16]. Worker ants are infected during foraging by spores that attach to the cuticle. The fungus is an obligate, directly transmitted parasite that requires ants for reproduction. Germination and subsequent penetration of the cuticle lead to rapidly progressing infections inside the host body [17,18], but fungal reproduction is only possible after the growth of a large stalk from the back of the ant’s head followed by a propulsive release of spores from this fruiting body [16]. The fungus inevitably kills the ant and must do this outside the colony because ants quickly remove dead nest-mates [19], so that dying in the nest would not allow sufficient time for stalk development and spore release [20]. This host death as a developmental necessity implies that Ophiocordyceps infections would also match the functional definition of being a parasitoid [21]. While the Ophiocordyceps clade has a global distribu- tion local interactions tend to be highly specific with highly stereotyped host behaviors [16,22]. Ants of the tribe Camponotinii (Camponotus, Polyrhachis and Echi- nopla) are known to leave their nest to bite into leaves before dying from infections with a representative of the species complex Ophiocordyceps unilateralis sensu lato [16]. (See taxonomic note in Methods). A recent study in a Thai rainforest showed that leaf biting behavior by infected workers of this ant species was adaptive for the fungus because it secures a stable microclimatic niche for the post mortem development of the stalk and the subse- quent release of spores [20]. In this intensively studied population infected worker ants leave their colony in the dry, hot canopy and descend to the humid understory where they appear to actively select leaves of saplings ca. 25 +/- 3 cm above the soil surface [20]. These parasite manipulated ants always bite into abaxial leaf veins and not the laminar blade, edge or upper surface (adaxial). They also predominantly die in areas where the cadavers of previously manipulated ants are already abundant leading to graveyards where local densities of ants killed by the fungus may exceed 25/m2 [23]. Graveyards of Ophiocordyceps infected ants have also been reported from other continents [24,25]. The healthy ants, though nesting in the high canopy, do periodically walk in the understory leading to new infections [23]. Behavioral manipulation of worker ants by these fungi creates zombie ants [2,4]. Once infected ants exit their colony to die they have no further fitness gains through their own actions (being sterile workers they only have inclusive fitness via helping nest-mates). In fact, by dying within the foraging area of their own colony their behavior may still reduce inclusive fitness [26-28]. The term zombie ants underlines that, while the manipulated individual may look like an ant, it represents a fungal genome expressing fungal behavior through the body of an ant. Using the explicit ‘parasite’s eye view’ framework out- lined above, we set out to test two hypotheses. First, we hypothesized that pre-biting behavior may have an important function to help positioning dying ants in death biting habitats that would be optimal for subse- quent fungal reproduction. Second, we hypothesized that the death grip requires changes in the mandibular muscles to transform functional mandibles into death grip lock-jaws to secure that dead ants become perma- nently fixed to leaves against the force of gravity. Results Pre-biting behavior The ant species that is the primary host of Ophiocordy- ceps unilateralis s.l. at our field site is Camponotus leo- nardi [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 vegeta- tion, 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 emer- gence 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 ulti- mately 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 Hughes et al. BMC Ecology 2011, 11:13 http://www.biomedcentral.com/1472-6785/11/13 Page 2 of 10 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, suggest- ing 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 walk- ing 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 observa- tions, the collected trail ants were maintained singly with- out 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 tra- versed 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 Figure 1 Zombie ant behavior. Focal animal observation periodogram of ants infected by Ophiocordyceps unilateralis s.l. The blue horizontal bars mark the observation period, the red triangles mark moment of biting, the vertical bars mark spasm events and the grey diamonds the falling off events. For four individuals that belong to focal animals 1-9 only the biting time was recorded. The biting time was recorded for 16 ants but only 15 triangles are visible as two ants bite at exactly the same time (12:05). Inset picture shows a dead ant on a leaf with the fungal stalk and spore body that emerged from the head. Figure 2 Synchronized manipulation of ants by fungi. A sun position chart of the death grip. Solar altitude is represented by the yellow bars and plotted against the y-axis and the biting times are the red circles and plotted on the x-axis in solar time (this is true local time accounting for longitude and different from Time in Figure 1). The red circles are stacked to prevent overlapping. At 11:47 two ants bite so only 15 circles are visible though 16 ants were recorded. Hughes et al. BMC Ecology 2011, 11:13 http://www.biomedcentral.com/1472-6785/11/13 Page 3 of 10 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), sug- gesting 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 hap- pened it was due to disturbance (two cases, # 15 and 18 Figure 3 Heads of manipulated ants colonized by fungi. A (top panel) is a light micrograph (LM) saggital section through the head of an O. unilateralis s.l infected ant that was biting a leaf at the moment of fixation (i.e. alive). The small grey blobs are fungal hyphal bodies that fill the head and mandibles. Note the spacing between the muscle fibers. The insect shows a close up of hyphal bodies around the post-pharyngeal gland (PPG). B is the brain, Mu, Muscles and Cu is cuticle. B) is a LM of healthy muscle and C) is a LM of muscle from a behaviorally manipulated ant that was biting a leaf and alive when removed for fixation. The small blobs between the fibers are fungal cells. Hughes et al. BMC Ecology 2011, 11:13 http://www.biomedcentral.com/1472-6785/11/13 Page 4 of 10 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 mus- cle 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. Mandibu- lar muscle fibers, which normally attach to the head capsule, often appeared to have become detached (Fig- ure 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 appen- dages are pulled across the maxillae to be cleaned. At the sub-cellular level (as seen with TEM) the mus- cles 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) fila- ments which attach during cross-bridge cycling leading to muscle shortening. To achieve contraction mitochon- dria 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 reticu- lum 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 trans- mission electron micrographs showed a distinct atrophy in the muscles of infected ants. Despite the apparent atrophy of muscles the behavio- rally manipulated ants were able to exert considerable force. We removed 29 dead ants from divers