Parasite Attraction to Host Cues

Patterns of attraction of Escovopsis towards cultivars in experimental bioassays are consistent with patterns of host use in nature. In bioassays, all three Escovopsis spp. were more likely to be attracted to isolates of their typical cultivar hosts than to distantly related cultivars. Furthermore, more specialized pathogens had more specialized responses: yellow Escovopsis, which attacks only cultivar A in nature, was more rapidly attracted to cultivar A over cultivar B, whereas brown Escovopsis, which naturally attacks both of these cultivar types, did not show preferential attraction. Upon arriving at susceptible cultivars, Escovopsis quickly overgrew the cultivar isolates, established infection, and presumably degraded the host [17].

Paralleling the case of Escovopsis, pre-contact attraction to host cues is critical in the life-history of many symbionts, and distantly related parasites use chemotaxis to locate their hosts. Nematodes, for example, are chemotactically attracted to their plant hosts [7], and pathogenic bacteria are attracted to substances released by their host fungi [21, 22]. Similarly, many pathogenic fungi and fungus-like organisms have zoospores, asexual spores with flagella, which swim towards a wide range of chemicals produced by their plant and fungal hosts [10]. Besides Escovopsis, few fungi without zoospores are known to exhibit specialized, directed growth towards their hosts [23, 24]. One interesting case is arbuscular mycorrhizal fungi, mutualistic associates of plant roots, which are known to be stimulated to grow specifically towards volatile cues produced by host roots [25, 26]. Such attraction to host cues is advantageous because it allows both parasites and mutualists to establish contact efficiently with their hosts rather than to depend on random movements to commence association.

Host Defense against Parasites

In general, host defense is widespread because it benefits any host to defend itself against virulent pathogens. Here, microbial bioassays suggest that cultivars can inhibit many Escovopsis isolates, which may prevent the establishment of infection by these pathogens in nature. In most bioassays, cultivar C inhibited yellow and brown, but not pink Escovopsis, whereas cultivars A and B often inhibited pink Escovopsis, but rarely inhibited yellow or brown Escovopsis. Therefore, like Escovopsis' attraction to cultivars, patterns of cultivar defense parallel Escovopsis–cultivar associations in nature and support the hypothesis that cultivar defenses maintain narrow Escovopsis host ranges and prevent host switching.

Genotype–Genotype Interactions

There were exceptions in which cultivars did not inhibit the pathogens to which they are not naturally associated. In five out of 16 choice bioassays and in 15 out of 100 no-choice bioassays challenging a yellow Escovopsis isolate against a cultivar-C isolate, the pathogen was able to overcome host defenses and establish infection. These cases suggest that yellow Escovopsis could potentially switch to this novel host.

Correlation analyses between genetic relatedness and outcomes of no-choice bioassays between yellow Escovopsis and cultivar C suggest genotype–genotype specificity in this non-host interaction. Genetically similar cultivar C strains are more likely to inhibit the same yellow Escovopsis strains than genetically dissimilar cultivars, and genetically similar Escovopsis are more likely to be inhibited by the same cultivar strains. Therefore, whether a cultivar strain can successfully suppress an Escovopsis strain appears dependent on the genotype combination. Though correlations between genetic relatedness and patterns of attraction were not significant at the p < 0.05 level, there may also be a biologically significant association between attraction patterns and genotype.

Two major models describe genetic interactions between hosts and parasites: the matching-alleles model and the gene-for-gene model. Under the matching-alleles model, a parasite's infectivity genotype must exactly match a host's susceptibility genotype in order to establish infection, and all parasites are equally successful when paired with their appropriate host. Under the gene-for-gene model, some parasites are more effective than others, and some hosts are more resistant than others but, owing to associated costs, no genotype spreads to fixation. Biological systems may incorporate both of these dynamics [27], and both could lead to correlation between genetic relatedness and infection. Here, no-choice-bioassay interactions between yellow Escovopsis and cultivar C are consistent with both models. Sometimes, only exact matches lead to infection, as expected under the matching-alleles model, and there are some yellow Escovopsis strains that infect all hosts and some cultivar C strains that are susceptible to all parasites, as expected under the gene-for-gene model. Such genotypic dynamics lead to the maintenance of polymorphism and the fluctuation of both host and parasite genotypes [28]. In this case, fluctuations are likely to be driven by interactions between parasites and their typical hosts, but in turn influence interactions between parasites and atypical hosts.

Conclusions

How may defense and attraction be coupled to maintain Escovopsis' host fidelity in nature? Though the mechanism by which Escovopsis is transmitted is not known, colonies with the different cultivar types (A, B, and C) can be found only centimeters apart in the field (Gerardo and Currie, unpublished data), increasing the possibility for transmission between hosts. If an Escovopsis strain is transmitted to a host with which it is not typically associated, however, the parasite would be able to overcome the cultivar's defenses and establish infection only if the right host and parasite genotypes came into contact. Upon reaching the proximity of a garden with the appropriate cultivar, Escovopsis would be able to spread quickly through the garden matrix as it is attracted to portions of the garden with the fungal cultivar. This process could make the establishment of infection more rapid and may prevent the successful suppression of the parasite by the ants, which have behaviors specific to the removal of Escovopsis [29], and also by filamentous bacteria on the ants, which are known to produce secondary metabolites that inhibit Escovopsis growth [30, 31].

Over evolutionary time, Escovopsis' host fidelity may facilitate cospeciation—the process by which interacting organisms speciate in tandem. Few studies have shown how adaptive evolution may drive cospeciation [12, 32]. In the attine ant–microbe symbiosis, both attraction to the host on the part of parasites, as well as host defenses, may limit host switching, reinforcing long-term host–parasite associations and thus facilitating cospeciation. Because these microbial symbionts coevolve with ants and with bacteria on the ants, these adaptations may also indirectly affect the speciation processes of these organisms. Certainly, other adaptations, including ant behaviors, modes of transmission, bacteria-derived antibiotics, and fungal growth properties, are likely to influence the probability of persisting on novel hosts. Future studies should explore the suite of adaptations involved in maintaining specificity of all four symbionts and the pace at which chemical and behavioral adaptations have coevolved over the ancient history of this complex host–pathogen association.

Collections.

Fungi were cultured from gardens of fungus-growing ants in the genus Apterostigma in Panama and Costa Rica following procedures described in [20]. Yellow-spored and brown-spored Escovopsis isolates (Figure 1) were from A. dentigerum colonies, which raise cultivar A, while all pink-spored Escovopsis isolates were from A. auriculatum colonies, which raise cultivar C (Figure 1). Besides differences in the color of their conidia (yellow, brown, and pink), the three Escovopsis types also have distinct micromorphological differences of their conidiophores (Currie, unpublished data) and fall into three distinct phylogenetic groups (Gerardo et al., unpublished data). No two isolates of the same morphological type were sampled from the same colony.

Cultivar-A isolates were from A. dentigerum colonies; these cultivars fall into the “G2-clade” in [14]. Cultivar-B isolates were from A. cf. manni colonies; these cultivars fall into the “G4-clade” in [33]. Cultivar-C isolates were from A. auriculatum colonies; these cultivars fall into the “G3-clade” in [14] and, specifically, into “Clade-1” of the G3-clade in [34]. Cultivar-A and cultivar-B isolates are in the family Pterulaceae, and cultivar C isolates are in the family Lepiotaceae [33].

Fungal-choice bioassays.

For bioassays with yellow Escovopsis, agar in 14-cm petri dishes filled with 50 ml of PDA plus antibiotics (potato dextrose agar with 50 mg/l each of penicillin and streptomycin) was cut out to leave four 4-cm-wide tracks (Figure 2A). For each plate, each track was randomly assigned to one of four treatments: control (no cultivar), cultivar A, cultivar B, or cultivar C. One of eight cultivar-A isolates, one of eight cultivar-B isolates, and one of four cultivar-C isolates was randomly assigned to each plate. Plates were inoculated with ≈6-mm³ agar pieces covered with mycelium from the appropriate cultivar culture. After 1 wk, plates on which all three cultivar isolates had grown without contamination (27 out of 35) were further inoculated with a ≈6-mm³ agar piece with spores and mycelium of one of 12 randomly assigned Escovopsis isolates. Plates were photographed every 1–3 d for 3 mo and were monitored occasionally for several months thereafter. From photographs, we estimated the number of days (#days) that it took Escovopsis to reach the end of each track. We excluded data from 11 plates on which Escovopsis had not reached the end of the control track within 90 d, because it was unclear whether the cultivars and Escovopsis were still viable in these trials.

With data from the remaining 16 plates, we used a random-effects analysis of variance (PROC MIXED, SAS Institute, http://www.sas.com/service/techsup/faq/stat_proc/mixeproc.html) to compare #days (log-transformed) to reach cultivar A, cultivar B, and the end of the control track, treating plate and Escovopsis isolate as random effects (in order to account for variation between plates and repetition of parasite isolates) and cultivar type as a fixed effect. We used log-likelihood ratio tests to confirm that there was no effect of random variables, and conducted pairwise, Bonferroni-corrected comparisons of least-squares means of the treatments (A, B, and control).

The fungal-choice bioassays with brown Escovopsis were similar, except that we inoculated 26 plates with one of eight cultivar-A, one of two cultivar-B, and one of six cultivar-C isolates and, 1 wk later, with one of ten brown Escovopsis isolates. Data from four of these plates were excluded because the date at which Escovopsis reached at least one of the tracks could not be estimated accurately from the available photographs; in these cases, photographs were not taken regularly around the time that the parasite reached the track end. Because cultivar C inhibited Escovopsis growth on 32 out of the 38 final plates in the dataset, #days to cultivar C was not included in the above analysis of variance. Instead, for trials in which cultivar C was overgrown, combining data from both the brown and yellow Escovopsis trials, we used a Wilcoxan rank sum test to compare #days to control versus #days to cultivar C.

No-choice bioassay I.

For each no-choice bioassay, we placed a single isolate of cultivar near the edge of a 9-cm petri dish with PDA plus antibiotics, as described. After 1 wk, we inoculated the center of each plate with a single Escovopsis isolate. Each of nine cultivar isolates (three A, three B, and three C isolates) was interacted with each of the same nine Escovopsis isolates (three yellow, three brown, and three pink isolates) for a total of 81 bioassays. Bioassays were monitored for up to 2 mo. Interactions were scored for the presence/absence of inhibition and the presence/absence of attraction.

No-choice bioassay II.

In a second no-choice experiment, following the same protocol outlined above, ten cultivar-C isolates were interacted with each of ten yellow Escovopsis isolates for a total of 100 bioassays. We then used amplified fragment-length polymorphisms (AFLPs) to generate genotype fingerprints of the cultivar strains, following the protocol outlined in [20]. To obtain fingerprints of the same level of genetic variability for Escovopsis, we sequenced 552 bp of elongation factor 1-alpha, using primers 3F and 5R, following the protocol described in [20]. With these data, using PAUP* (version 4b10 [35]), we constructed two genetic-distance matrices—a Nei–Li distance matrix for the ten experimental cultivar strains and a maximum-likelihood distance matrix for the ten experimental Escovopsis strains ( Figure S1). For maximum-likelihood distances, we used the TrN + Γ + PINVAR model of evolution, where TrN is the Tamura–Nei model of DNA evolution, and PINVAR is the proportion of invariant sites as determined via Modeltest (version 3.06, [36]).

Four interaction-distance matrices were constructed ( Figure S1). The first matrix consisted of the inhibition distances between each pair of the ten cultivar C strains, where each inhibition distance ranged from 0 to 1 and increased by 0.1 for each case in which the two cultivar strains had a different inhibition result with the same Escovopsis strain (i.e., one cultivar inhibited the Escovopsis strain while the other did not). A second matrix consisted of Escovopsis inhibition distances; each inhibition distance ranged from 0 to 1 and increased by 0.1 for each case in which the two Escovopsis strains had a different inhibition result with the same cultivar strain (i.e., one Escovopsis strain was inhibited while the other was not). The third and fourth matrices were comprised of cultivar and Escovopsis attraction distances, which were determined in a similar manner to the inhibition distances. We then used ZT [37] to conduct Mantel tests to examine the correlation between matrices of: (1) cultivar genetic distances and cultivar inhibition distances, (2) Escovopsis genetic distances and Escovopsis inhibition distances, (3) cultivar genetic distances and cultivar attraction distances, and (4) Escovopsis genetic distances and Escovopsis attraction distances.

Accession Numbers

The GenBank ( http://www.ncbi.nlm.nih.gov) accession numbers for the Escovopsis sequences discussed in this study are DQ415661–DQ415670.