Computational protocol: Interaction specificity between leaf-cutting ants and vertically transmitted Pseudonocardia bacteria

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[…] Following the 3 days of behavioral observations after Escovopsis treatment the subcolony fungus garden fragments were replaced with 2 g of fresh fungus garden including ca. 25 medium workers and 50 small workers from the same source colony that experimental workers eclosed in, ensuring that the subcolonies had fresh fungus garden and work force to maintain the fungal cultivar. The bacterial cover of each marked worker was estimated two weeks after Escovopsis exposure as described above. Subsequently the ants were frozen at −20°C for PCR identification of the Pseudonocardia phylotypes.From each of the subcolonies, one individual ant was used for analysis of Pseudonocardia phylotype identity. Samples from colonies Ae.227 and Ae.266 were also included for identification of their native Pseudonocardia phylotype, to allow comparison of our present results with the earlier cross-fostering experiment []. The propleural plates were dissected off the ants under a stereomicroscope following the procedure outlined in Andersen et al. [] and DNA extracted with the DNEasy Blood and Tissue kit following the manufacturer’s instructions (Invitrogen, Hilden, Germany). Part of the elongation factor EF-1α gene was amplified with the Pseudonocardia specific primers 52 F and 920R [] in 20 μL PCR reactions with the AmpliTaq Gold kit (Applied Biosciences, New Jersey, USA) at the conditions: 95°C for 4 min followed by 40 cycles of 95°C for 30 s, 62°C for 50 s and 72°C for 2 min and final extension at 72°C for 10 min. PCR products were purified with an MSB Spin PCRapace kit (Invitek, Berlin, Germany) and sequenced by Eurofins MWG Operon (Ebersberg, Germany). Successful PCR amplification was achieved for all but three experimental subcolonies that either had a very low bacterial cover or failed for unknown reasons. The sequences were trimmed and compared in Sequencher 4.7 and the consensus sequences of both phylotypes compared to known sequences with a NCBI GenBank BLAST search. These were 99% similar (Ps1, [GenBank: DQ098127]) and identical (Ps2, [GenBank: DQ098133]) to sequences of bacteria sampled in the same area by Poulsen et al. [] and 95% similar to each other. [...] Bacterial cover was estimated for each individual at each time step (t), and a logistic growth equation (Cover = KP0ert / K + P0(ert-1)) fitted to the data, with K (the “carrying capacity”) set to the maximum cover (scale 12), and the value of r (the intrinsic rate of increase in bacterial cover) estimated for each individual using iterative least squares fitting executed in the solver add-in of Microsoft Excel 2011. The single value of P0 (the bacterial cover at day 0) that provided the best fit to the data across all individuals was estimated iteratively to be 2.67 × 10−4. Bacterial cover on Day 19 was only estimated for 21 of the 32 subcolonies, as the remaining were already treated with Escovopsis at this stage, but for these subcolonies the bacterial cover was not significantly different from the cover on Day 15 (Wilcoxon Signed-Rank test, S = −7, p = 0.436; Figure A).Growth rates were Box-Cox transformed to maximize normality and homogeneity of variance for analysis. The rates of increase in bacterial cover and the final cover two weeks after infection were analyzed with a two-way ANOVA to test whether Pseudonocardia phylotype of the foster garden and nurses, the native phylotype that the pupae should have been inoculated with, and the interaction between the two affected cuticular growth. The ID of the source colony of the fungus gardens and the pupae was included as a nested variable with the replicate subcolonies nested within. Analyses were carried out using JMP 10.0.2 for Macintosh.The achieved power of the analysis was analyzed post-hoc with G*Power 3.1.4 [] to see how likely we were to detect a Pseudonocardia phylotype effect given the substantial variation between colonies and our sample sizes. This showed that an effect size (f) of around 0.3 could be detected with a power of 80%, which corresponds to a medium effect size in Cohen’s classification []. Substantial differences in bacterial growth rate between treatments should thus have been detectable with the sample size used in our study, suggesting that any Pseudonocardia phylotype-specific effects are generally smaller than colony-specific effects.The final bacterial cover on ants two weeks after Escovopsis exposure was analyzed with an ordinal logistic model using maximum likelihood in JMP 10.0.2 for Macintosh, to test whether the Pseudonocardia phylotype of the foster garden and nurses, the native phylotype that the pupae should have been inoculated with, and the interaction between the two affected it. Three subcolonies lost more than half of their fungus garden in the course of the experiment, but the decreased garden mass did not affect the growth of Pseudonocardia cover (One-Way ANOVA, F1,125 = 0.142, p = 0.707). However, they maintained a high bacterial cover rather than showing the characteristic decrease of workers from the other subcolonies, and so had a significantly higher cover two weeks after infection (Ordinal logistic model, Likelihood-Ratio χ2 = 6.50, d.f. = 1, p = 0.011). As a result, these subcolonies were excluded from the analysis of final bacterial cover, and from the behavioral analyses to avoid the possibility that reduced garden mass could affect the results. […]

Pipeline specifications

Software tools Sequencher, G*Power
Applications Miscellaneous, Phylogenetics
Organisms Acromyrmex echinatior