Manipulated dead ant collection and species verification

Previous work on Ophiocordyceps in ants has focused on tropical habitats with the consensus that these fungi are uncommon in temperate biomes [26]. However, we discovered a large population of Carpenter ants infected with O. unilateralis s.l. in Donald’s County, South Carolina, USA. During our first field survey in 2009 we identified and individually marked 264 fungal killed Carpenter ants (genus Camponotus) at one site: 175 C. castaneus (Figure 1a) and 82 C. americanus (Figure 1b). All ants were attached to twigs from a number of tree species. Position of the death grip was highly stereotyped with 99% of ants attached to the adaxial (lower) side of twigs. Ants were never recorded attached to leaves, which occurs for infected Carpenter ants in tropical habitats [20]. Similar patterns were found in the following years (2010–2013) at this and 5 other sites.

Figure 1 Natural and lab infections with O. unilateralis s.l. . (A-B) C. castaneus (A) and C. americanus (B) infected with O. unilateralis s.l. collected in Donalds, SC. (C) O. unilateralis s.l. culture isolated from an infected C. castaneus specimen. (D-E) Manipulated C. castaneus (D) and C. americanus (E) upon infection with O. unilateralis s.l. in the lab. Full size image

We isolated O. unilateralis s.l. from a freshly manipulated and killed C. castaneus cadaver (Figure 1c). Species verification was done through SSU (small subunit ribosomal RNA gene) sequencing. Blasting the obtained PCR fragment sequence (KJ769099) against all fungi in the NCBI database resulted in a 97% identity with both O. unilateralis strain OSC 128574 (DQ522554 [32]) and the very closely related Ophiocordyceps pulvinata voucher TNS-F-30044 (GU904208 [24]), verifying that we successfully isolated O. unilateralis s.l..

Ant infections leading towards species-specific behavioral manipulation

In this study, we addressed if our O. unilateralis s.l. species could control the behavior of ants that are not normally found infected in nature. We chose C. pennsylvanicus and F. dolosa, which we never found infected at our site despite them occurring sympatrically and 1,750 person hours of searching over four years. C. pennsylvanicus is within the same genus as the two encountered hosts, C. castaneus and C. americanus, and is very abundant throughout the East Coast of the USA [33] including South Carolina [34]. F. dolosa is an ecologically similar species to C. castaneus and C. americanus occurring in mixed woodlands with colonies in the soil [35]. The genus Formica has never been recorded as a host to O. unilateralis s.l.[26],[36],[37]. The two naturally infected ant species were compared to the two naturally uninfected species using infection studies. Since the insect cuticle is a highly heterogeneous structure that varies between species, it is likely to affect the ability of fungal spores to adhere and penetrate the cuticle needed to establish infection [38]. Therefore, we bypassed such external barriers by directly injecting fungal cells into the ants. For each species, 3 replicates of 40 worker ants were placed in a cage containing branches that served as a biting platform and a darkened nest area. Per replicate, 10 ants were injected with fungal cells, 10 were sham treated, and 20 were left untreated. Cages were kept under strict twenty-four hour light and temperature cycles since ant behavior is highly dependent on genes oscillating with circadian rhythms [39]–[41]. Furthermore, field studies on O. unilateralis s.l. infected Camponotus leonardi in Thailand, showed synchronization of manipulated biting behavior at solar noon [19]. Next to that, the convergently evolved entomophthoralean fungi are known to cause comparable behavioral manipulations in a range of arthropods (including ants), followed by death and sporulation which also happens at distinct times of day [42]–[45]. Observations were made 5 times per day for 28 continuous days following infection, recording behavior and time of death for each of the 480 individually marked ants. This set up resulted in the successful reconstruction of behavioral manipulation of ant behavior by a fungal parasite under controlled laboratory conditions.

The three Camponotus species infected with O. unilateralis s.l. had similar survival probabilities (p = 0.475, log-rank test of Kaplan-Meier survival probabilities, Figure 2a). Sham treated and untreated ants for all three species had a significantly higher survival probability than infected individuals (p = 0.00, log-rank test of Kaplan-Meier survival probabilities, Figure 2a). Formica dolosa reacted adversely to the injection procedure and was excluded from the analysis. Following death, ant cadavers were monitored for fungal growth emerging from them, which is necessary for parasite transmission. For all three Camponotus species no fungal growth was observed upon death <9 days post infection. Fungal growth did emerge from C. castaneus and C. americanus individuals that died >/=9 days post infection (Figure 2a and Additional file 1a,b). We never observed fungal hyphae emerging from the non-target host cadavers of C. pennsylvanicus (Figure 2a). However, fungal blastospores were found inside the bodies of C. pennsylvanicus (Additional file 1c,d), demonstrating that the cause of death in these ants was likely due to fungal growth.

Figure 2 Survival, fungal growth, and behavioral manipulation of three Camponotus species infected with O. unilateralis s.l. . (A) Kaplan-Meier survival curve for 3 different Camponotus species infected with O. unilateralis s.l.. The thickened lines represent the time period in which fungal growth was observed. The grey box indicates days post-infection during which behavioral manipulation was observed. (B) Mean proportion of observations outside for each species-treatment combination (data presented as mean +/− SD, P<0.001 Tukey post-hoc on two-way ANOVA). Full size image

In addition, we recorded ant behavior. Infected ants were observed in the foraging arena significantly more often than their healthy and sham-treated nest mates (Tukey post hoc on two-way ANOVA, adjusted p < 0.001 Figure 2b). Only the two species of ants known to be naturally infected (C. castaneus and C. americanus) were manipulated to die biting (Figures 1d,e, 2a and Additional files 2 and 3), while C. pennsylvanicus was never found biting twigs prior to death. Similar to fungal growth, there seems to be a critical period for manipulated biting behavior because we only observed successful manipulation between 14 to 22 days post infection. This implies a parasitic growth phase within the host is needed and that premature death of the host prevents successful fungal development. We suggest that the complex nature of manipulation partially explains why such a long incubation period inside the host is required.

Additional file 2: Manipulated biting behavior in C. castaneus. Video recording of manipulated biting behavior seen in C. castaneus.(MOV 11 MB)

Additional file 3: Manipulated biting behavior in C. americanus. Video recording of manipulated biting behavior seen in C. americanus.(MOV 7 MB)

Metabolomics of species-specific ex vivo ant brain–O. unilateralis s.l.interactions

Only the two naturally occurring hosts of O. unilateralis s.l. could be manipulated despite the ability of the fungus to establish inside all three Camponotus species. The lack of manipulation in the non-target ant species, despite the ability of the fungus to kill it suggests that this requires additional factors. The complexity of the manipulated behavior suggests a specific reaction to the host’s CNS is required. This led us to ask if O. unilateralis s.l. secretes a different array of metabolites (i.e. displays a heterogeneous secretome) when presented with ant brains of the four different species used in this study. In this experiment, F. dolosa has been included again as it allowed us to ask how O. unilateralis s.l. would react to ant brains from a different genus. We used a novel protocol that allows the investigation of the secretome of fungal entomopathogens as a reaction to specific insect tissues kept alive ex vivo[31]. Through quadrupole time-of-flight mass spectrometry, we measured the unique mass-to-charge/retention time pairs (features) that were significantly enriched in samples where O. unilateralis s.l. was grown in the presence of ant brains. Biological replicates for the fungal-brain interaction samples and the various controls (see Methods section) were run together in a randomized order. Within the chosen window of 0.9 and 15 minutes and m/z between 100 and 1100, 37,921 unique ion features were extracted from the raw data (Additional file 4a and [46]).

We performed a principal component analysis and discriminant analysis (PCA-DA, in which discriminating components are calculated with foreknowledge of the samples) on all ion features. This resulted in the clustering of the biological replicates for each species interaction suggesting that, depending on the ant species brains it was presented with, the secretome of O. unilateralis s.l. differed (Figure 3a). However, control samples representing the different species’ brains, could also be separated by PCA-DA (Figure 3b). It is therefore possible that the clustering seen for the brain-fungal interactions might be partially due to the brain tissues. This implies that directly comparing O. unilateralis s.l. grown beside the brain of one ant species with it grown beside the brain of another is not the best approach when the aim is to specifically study the fungal secretome. Such an approach would lead to the complication of extracting the differences between different species brain tissues from the data set together with the compounds of interest secreted by the fungus as a reaction to those different tissues. Therefore, we performed statistical analyses within a species to rank enriched ion features involved in fungus-brain according to significance, prior to performing an indirect comparison between the species using those ranked ions. Separate PCA-DAs for each of the four species tested resulted in a separate clustering of O. unilateralis s.l.–ant brain interaction samples and their corresponding species-specific controls (Figure 3c-f), showing that O. unilateralis s.l. reacts to brain tissue through secretion. To rank the ion features enriched as a result of O. unilateralis s.l.-ant brain interaction, a t-test for each species was performed in which this interaction was compared to the various controls. Only those ion features that were found to be enriched with a p-value of p < 0.01 were used in the indirect, between species, comparison. We found 170, 86, 104 and 206 significantly enriched molecular weight/retention time peaks for O. unilateralis s.l. growth beside C. castaneus, C. americanus, C. pennsylvanicus and F. dolosa brains respectively (for feature IDs see Additional file 5). We compared the enriched ion features found for each species interaction, which showed that most of the ion features (between 69% and 85%) were only significantly enriched in one of the four parasite-ant brain interactions (Figure 4a). Our study of the O. unilateralis s.l. secretome thus indicates that this fungus reacts heterogeneously to brains from the four ant species we studied by secreting a largely different array of metabolites.

Figure 3 PCA-DA analyses to determine the heterogeneity of O. unilateralis s.l. on different ant species’ brains. (A) PCA-DA plot showing the clustering of O. unilateralis s.l. secretion in the presence of different ant species’ brains kept ex vivo in Schneider’s insect medium and the medium without ant brains. (B) PCA-DA plot showing the clustering of different ant species’ brains kept ex vivo and the medium by itself without fungal growth that served as controls in this study. (C-F) PCA-DA plots showing the clustering per species of O. unilateralis s.l. secretion in the presence of ant brains of that species versus secretion in the medium without ant brains, ant brains kept ex vivo without fungal growth and the Schneider’s insect medium by itself. Full size image

Figure 4 Heterogeneous metabolite secretion by O. unilateralis s.l. on four ant species’ brains. (A) Venn-diagram comparing all ion features found to be significantly (P < 0.01) enriched in the medium of samples in which O.unilateralis s.l. was grown next to ex vivo kept ant brains of the species C. castaneus, C. americanus, C. pennsylvanicus and F. dolosa. (B) Bar chart visualizing how the ions, that were found to be significantly enriched (P < 0.01) due to O. unilateralis s.l.-C. castaneus brain interactions across two independent studies, overlap with ions enriched in interactions with other species’ brains. Full size image

To investigate if these results could be (partly) due to false positives and if our findings hold when the analysis is performed across two independently set up experiments, we repeated the part in which O. unilateralis s.l. was grown beside brains of C. castaneus, the host species from which we isolated it. In addition, in this set up, the fungus was also grown beside the mandibular muscles of its host. We included this tissue in the analysis to exclude the features that are enriched because of the interaction with ant tissue in general, but are not necessarily brain tissue specific. This resulted in 41,254 unique ion features (Additional file 4b and [46]), which we analyzed using PCA-DA plots that showed clustering of the different biological sample types, as seen before (Additional file 6a-c). Running the samples at the same time and the use of an internal standard in both experiments allowed the comparison of this experiment with the former one. The samples of this second experiment were analyzed together with the C. castaneus related samples of the first one (Additional file 4c, Additional file 6d). To rank the ion features that were significantly enriched as a reaction to brain tissue, again a t-test was performed in which the fungus-brain interaction was compared to the various controls. Choosing again a cut off of p < 0.01 we found 258 significantly enriched ion features within the secretome of O. unilateralis s.l. grown on C. castaneus brains (Additional file 7a). Comparing these enriched ion features with those found to be enriched for O. unilateralis s.l.-C. castaneus brain interactions in first analysis described above, 56 were found to be in common (listed in Additional file 8). The discovery of these significantly enriched ion features across two independent experiments suggests that these are biologically relevant and not false positives due to the experimental set up or data mining. We compared these 56 ion features that O. unilateralis s.l. produced in the presence of C. castaneus brains again with the ion features found to be enriched for O. unilateralis s.l. growth in the presence of other ant species brains. Of these ion features, 73% were only significantly enriched in the presence of C. castaneus brains (Figure 4b). This more conservative analysis of the data thus resulted in the same conclusion that O. unilateralis s.l. secretes a specific set of metabolites depending on the ant brain it encounters. In addition to providing insight into the patterns of specificity of organisms evolved to control brains, these experiments resulted in candidate compounds that might be involved in establishing behavioral manipulation.

Identification of candidate metabolites involved in brain manipulation

Our attempt to identify metabolites focused on the ion features enriched in the secretome of O. unilateralis s.l. in the presence of brains of its natural host C. castaneus. We found 56 ion features across two experiments (Additional file 8). In an attempt to identify these we compared their MS/MS product ion mass spectra with those of a similar m/z value in the METLIN database [47]. A present roadblock is however that metabolite databases do not yet hold an extensive amount of mass spectra and do not cover all eukaryote species to the same extent. Therefore, in most of the cases where a MS/MS product ion scan was generated, no match was found. This of course results in limitations that are inherent to the use of metabolomics in a study like the one presented here, but with more data available, this will improve over time. Despite this, we did manage to putatively identify one of the candidate ion features to be guanidinobutyric acid (GBA, m/z 146.0914 at 1.08 min., 2.6 fold higher in the secretome of O. unilateralis s.l. grown in the presence of C. castaneus brains, Additional file 7a). This identification was verified by comparing the retention time and product ion MS/MS spectra obtained from our samples with an authentic standard (CAS 463-00-3, Sigma Aldrich; Additional file 9a). GBA is involved in the transport of compounds such as creatine and guanidinoacidic acid (GAA) across the blood–brain barrier [48] and known to be involved in epileptic discharges and convulsions in rodents [49]. Altered levels of creatine and GAA have been shown to cause neurological disorders [50]. GBA has also previously been isolated from the fungus Trogia venenata, which has been implicated in causing sudden deaths in Yunnan, China [51].

To search for additional compounds we adopted a less stringent but still significant cut off value of p < 0.05 for our ranking analysis across the two experiments. This returned an additional 1038 ion features, which still comprises only the top 2.6% of all ion features from the secretome of O. unilateralis s.l. grown beside the brains of C. castaneus (Additional file 7b). Among these features we putatively identified a sphingosine (m/z 300.2891 at 12.96 min., 2.6 fold higher in the secretome of O. unilateralis s.l. grown in the presence of C. castaneus brains, Additional file 7b). The identified metabolite was verified by comparing the retention time and product ion mass spectrum to that of an authentic standard of L-threo-sphingosine (CAS 25695-95-8, Cayman Chemical; Additional file 9b). Sphingosines are part of sphingolipid metabolism, which affects all types of cell regulation [52],[53]. Defects can lead to cancers [54] and neurological syndromes [55]. The secretion of fungal derived sphingosines have not been reported to this date, but several different fumonisins, that mimic compounds involved in sphingolipid metabolism, have been [56]. These compounds are produced by several plant pathogenic Fusarium species growing on cereals causing leukoencephalomalacia (‘hole in the head disease’) in life stock being fed with infected crops [57]. We examined our metabolomics dataset for these fumonisins but did not find any.

We identified two compounds that were enriched in the secretome of O. unilateralis s.l. grown beside C. castaneus brains. Since these compounds (GBA and sphingosine) have known neurological effects on mammals [49],[50],[55] we tested their effect on ants. We injected serial dilutions of the authentic standards of GBA and L-threo-sphingosine separately and in combination into ants of the species C. castaneus. This however did not result in behavioral effects similar to those induced by O. unilateralis s.l. infection. It is likely that the injection of specific candidate metabolites is not biologically realistic because based on our profiling of the fungal secretome we expect that multiple compounds, and therefore mechanisms, act in concert. This is in line with the conclusions that can be drawn from studies in T. gondii[14]–[17], described in the Background section.