Microbial Community Structure of Leaf-Cutter Ant Fungus Gardens and Refuse Dumps

BACKGROUND: Leaf-cutter ants use fresh plant material to grow a mutualistic fungus that serves as the ants' primary food source. Within fungus gardens, various plant compounds are metabolized and transformed into nutrients suitable for ant consumption. This symbiotic association produces a large amount of refuse consisting primarily of partly degraded plant material. A leaf-cutter ant colony is thus divided into two spatially and chemically distinct environments that together represent a plant biomass degradation gradient. Little is known about the microbial community structure in gardens and dumps or variation between lab and field colonies. METHODOLOGY/PRINCIPAL FINDINGS: Using microbial membrane lipid analysis and a variety of community metrics, we assessed and compared the microbiota of fungus gardens and refuse dumps from both laboratory-maintained and field-collected colonies. We found that gardens contained a diverse and consistent community of microbes, dominated by Gram-negative bacteria, particularly gamma-Proteobacteria and Bacteroidetes. These findings were consistent across lab and field gardens, as well as host ant taxa. In contrast, dumps were enriched for Gram-positive and anaerobic bacteria. Broad-scale clustering analyses revealed that community relatedness between samples reflected system component (gardens/dumps) rather than colony source (lab/field). At finer scales samples clustered according to colony source. CONCLUSIONS/SIGNIFICANCE: Here we report the first comparative analysis of the microbiota from leaf-cutter ant colonies. Our work reveals the presence of two distinct communities: one in the fungus garden and the other in the refuse dump. Though we find some effect of colony source on community structure, our data indicate the presence of consistently associated microbes within gardens and dumps. Substrate composition and system component appear to be the most important factor in structuring the microbial communities. These results thus suggest that resident communities are shaped by the plant degradation gradient created by ant behavior, specifically their fungiculture and waste management

Unique Honey Bee (<i>Apis mellifera</i>) Hive Component-Based Communities as Detected by a Hybrid of Phospholipid Fatty-Acid and Fatty-Acid Methyl Ester Analyses

<div><p>Microbial communities (microbiomes) are associated with almost all metazoans, including the honey bee <i>Apis mellifera</i>. Honey bees are social insects, maintaining complex hive systems composed of a variety of integral components including bees, comb, propolis, honey, and stored pollen. Given that the different components within hives can be physically separated and are nutritionally variable, we hypothesize that unique microbial communities may occur within the different microenvironments of honey bee colonies. To explore this hypothesis and to provide further insights into the microbiome of honey bees, we use a hybrid of fatty acid methyl ester (FAME) and phospholipid-derived fatty acid (PLFA) analysis to produce broad, lipid-based microbial community profiles of stored pollen, adults, pupae, honey, empty comb, and propolis for 11 honey bee hives. Averaging component lipid profiles by hive, we show that, in decreasing order, lipid markers representing fungi, Gram-negative bacteria, and Gram-positive bacteria have the highest relative abundances within honey bee colonies. Our lipid profiles reveal the presence of viable microbial communities in each of the six hive components sampled, with overall microbial community richness varying from lowest to highest in honey, comb, pupae, pollen, adults and propolis, respectively. Finally, microbial community lipid profiles were more similar when compared by component than by hive, location, or sampling year. Specifically, we found that individual hive components typically exhibited several dominant lipids and that these dominant lipids differ between components. Principal component and two-way clustering analyses both support significant grouping of lipids by hive component. Our findings indicate that in addition to the microbial communities present in individual workers, honey bee hives have resident microbial communities associated with different colony components.</p></div

Principal component analysis of lipid profile for honey bees and their hive components.

<p>Score plot shows variation in PLFA-FAME profiles by hive components. Lines drawn delineate sample aggregates. Inlay displays one propolis sample that skewed the plot. Main plot is redrawn without that data point. When produced by sample year, individual hive or location, no clustering is readily apparent.</p

Rarefaction and lipid accumulation curves for honey bees and their hive components.

<p><b>A.)</b> Observed and estimated curves resulting from community analyses of adults, honey, and pollen using <i>Mao Tau</i> and <i>Chao2</i> methods. <b>B.)</b> Observed and estimated curves from comb, pupae, and propolis community analyses. Dashed lines represent corresponding confidence intervals.</p

P-values from Tukey-Kramer HSD Pairwise Comparisons of Total Number of Lipids Detected in Each Component.

<p>NS—Not Significant</p><p>P-values from Tukey-Kramer HSD Pairwise Comparisons of Total Number of Lipids Detected in Each Component.</p

Lipid richness for honey bees and their hive components.

<p>Average number of individual lipids detected from each hive component. Error bars represent standard error of the mean. Letters represent Tukey-Kramer HSD pairwise comparisons, with those groups not possessing overlap in letters being significantly different. Those groups sharing letters do not significantly differ.</p

P-Values from Significant Tukey-Kramer HSD Pairwise Comparisons of Lipid Profiles By Component.

<p>*—Comparison Not Valid;</p><p>NS—Not Significant</p><p>P-Values from Significant Tukey-Kramer HSD Pairwise Comparisons of Lipid Profiles By Component.</p

P-Values from Tukey-Kramer HSD Pairwise Comparisons of Lipid Profiles by Year.

<p>*—Comparison Not Valid;</p><p>NS—Not Significant</p><p>P-Values from Tukey-Kramer HSD Pairwise Comparisons of Lipid Profiles by Year.</p

Average abundance of the most common lipids within honey bee colonies.

<p>Bar graph representing the average biomass of the most common lipids on a whole hive basis, generated by combining the individual hive components. Presented are those lipids that represent more than 1%. Error bars represent standard error of the mean. Abbreviations: Gram-negative (Gm-); Gram-positive (Gm+); γ-Proteobacteria (γ-proteo EV = <i>Enterobacter/Vibrio</i>); <i>Bacillus-Clostridium</i> group (BC); Saprotrophic Fungi (SP); Ectomycorrhizal fungi (Ecto).</p