Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia

Abstract

Background: Carpenter ants (genus Camponotus) are considered to be omnivores. Nonetheless, the genome sequence of Blochmannia floridanus, the obligate intracellular endosymbiont of Camponotus floridanus, suggests a function in nutritional upgrading of host resources by the bacterium. Thus, the strongly reduced genome of the endosymbiont retains genes for all subunits of a functional urease, as well as those for biosynthetic pathways for all but one (arginine) of the amino acids essential to the host.

Results: Nutritional upgrading by Blochmannia was tested in 90-day feeding experiments with brood-raising in worker-groups on chemically defined diets with and without essential amino acids and treated or not with antibiotics. Control groups were fed with cockroaches, honey water and Bhatkar agar. Worker-groups were provided with brood collected from the queenright mother-colonies (45 eggs and 45 first instar larvae each). Brood production did not differ significantly between groups of symbiotic workers on diets with and without essential amino acids. However, aposymbiotic worker groups raised significantly less brood on a diet lacking essential amino acids. Reduced brood production by aposymbiotic workers was compensated when those groups were provided with essential amino acids in their diet. Decrease of endosymbionts due to treatment with antibiotic was monitored by qRT-PCR and FISH after the 90-day experimental period. Urease function was confirmed by feeding experiments using 15N-labelled urea. GC-MS analysis of 15N-enrichment of free amino acids in workers revealed significant labelling of the non-essential amino acids alanine, glycine, aspartic acid, and glutamic acid, as well as of the essential amino acids methionine and phenylalanine.

Conclusion: Our results show that endosymbiotic Blochmannia nutritionally upgrade the diet of C. floridanus hosts to provide essential amino acids, and that it may also play a role in nitrogen recycling via its functional urease. Blochmannia may confer a significant fitness advantage via nutritional upgrading by enhancing competitive ability of Camponotus with other ant species lacking such an endosymbiont. Domestication of the endosymbiont may have facilitated the evolutionary success of the genus Camponotus.

Figure 1

Number of pupae raised by worker groups fed different diets over a 12-week period. The maximal possible number of pupae is 90. Differences across diets were significant (ANOVA: p < 0.001, F_5,42 = 13.338; see Results). Groups differing significantly from each other in Tukey HSD posthoc test are marked with different letters above box-plots. Medians are given in box-plots, and an outlier is marked with a circle. Diets: C = control (Bhatkar agar and honey water), A = artificial diet with essential amino acids, AR = artificial diet plus antibiotic Rifampicin, B = artificial diet without essential amino acids, BR = artificial diet without essential amino acids but with antibiotic Rifampicin, S = sucrose solution only (20% w/v).

Figure 2

Comparison of worker mortality across diets. Numbers of dead workers (of 150 individuals per group initially) after a 12-week experimental period; differences among diet categories were significant (ANOVA: p < 0.001, F_5,42 = 8.459; see Results). Groups that differed significantly from each other in Tukey HSD posthoc test are marked with different letter above box-plots. Diet A (artificial diet with essential AA) is intermediate between groups and not significantly different from these groups. Diets as in legend of Figure 1. Median is given as line in boxes, outliers are indicated by a circle.

Figure 3

Comparison across diet categories of numbers of brood items missing. Number of brood missing from a total of 90 eggs and first instar larvae distributed to each worker group over the 12-week experimental period. Differences across diets were significant (ANOVA: p < 0.001, F_5,42 = 6.287; see Results). Groups differing significantly from each other in a Tukey HSD posthoc test are marked with different letters above the boxes. Diet categories marked with two letters are intermediate and not significantly different from the respective groups. Diets as in legend of Figure 1. Lines within boxes represent medians, and an outlier is indicated by a circle.

Figure 4

Brood production across diet categories over time. Comparison of mean number of pupae produced over time for diets A (artificial diet with essential AA), diet AR (A plus antibiotic), diet B (A minus essential AA) and control (honey water, Bhatkar agar and cockroaches). Numbers of pupae produced are standardized relative values with minimum 0 when no pupae were present in the colony and the maximum number of pupae as 1. A logistic trend analysis revealed that worker groups fed diet AR were significantly slower in the production of pupae compared to groups on the three other diets.

Figure 5

Efficacy of antibiotic treatment verified by FISH. Blochmannia specific fluorescent in situ hybridisation (FISH) of bacteriocytes (Bc) in the midgut epithelium of: (A) and (B) an untreated worker of C. floridanus, and (C) and (D) a worker treated with the antibiotic rifampicin and fed an artificial diet containing all essential amino acids (fluorescence and phase contrast microscopy of the same region, respectively). The midguts of the untreated and the treated worker differ strongly in the number of visible (bacteria containing) bacteriocytes and the number of bacteria per cell. Epithelial gut tissue and other structures visible in phase contrast show no significant autofluorescence. Scale bar 10 μm.

Figure 6

Efficacy of antibiotic treatment measured by quantitive real-time PCR. Number of endosymbionts present in the workers after the experimental period measured as pg DNA of the amount of the housekeeping gene tufB. Workers differed significantly in their content of Blochmannia (ANOVA: Welch- test of square-root transformed data: p < 0.001, F_5,42 = 5.77). Groups differing significantly from each other in a Tamhane posthoc test are marked with different letters above the boxes. Diet categories marked with two letters are intermediate and not significantly different from the respective groups. Diets as in legend of Figure 1. Lines within boxes represent medians, and an outlier is indicated by a circle.