Fig 1.
Diagram of inoculation treatment methods.
(A) Brood frames from 3 colonies headed by unrelated single drone inseminated (SDI) queens were collected and 120 dark-eyed, tan pupae were extracted from each. (B) Pupae were placed into sterilized modular pupation plates, which were stored in individual sterilized Plexiglas cages in an incubator until bees eclosed. (C) After eclosion, bees were marked with a spot of paint indicative of their source colony and placed into 6 different treatment cages supplied with sterilized sucrose solution and sterilized artificial pollen patties. (D) Cages were supplemented with inoculum composed of a gut homogenate (Cages 1-4) or sterile PBS (Cages 5-6) for three days.
Table 1.
Relative abundance of each microbe in the gut microbial communities of treatment bees in each cage environment and genetic background (colony).
Table 2.
Absolute abundance of each microbe in the gut microbial communities of treatment bees in each cage environment and genetic background (colony).
Fig 2.
Host genetic background and social environment influenced gut microbial community structure.
(A) Inoculated bees from different genetic backgrounds and raised in different cage environments did not differ in gut microbiota alpha diversity. Shannon Alpha Diversity: Two-way ANOVA, Environment: F(3,48) = 1.217, p = 0.314, Genetic Background: F(2,48) = 1.639, p = 0.205, Environment*Genetic Background: F(6, 48) = 0.443, p = 0.846. (B) Inoculated bees from different genetic backgrounds differed in gut microbiota beta diversity. Two-way Permutation MANOVA using Aitchison Distance, Genetic Background: F(2,59) = 2.0523, R2 = 0.06392, p = 0.004; Environment: F(3,59) = 2.2910, R2 = 0.10703, p = 0.001; Genetic Background*Environment: F(6,59) = 0.8725, R2 = 0.08153, p = 0.809. n = 20 bees/colony (genetic background), 3 colonies. (C) Inoculated bees raised in different cage environments differed in gut microbiota beta diversity. Two-way Permutation MANOVA using Aitchison Distance, Genetic Background: F(2,59) = 2.0523, R2 = 0.06392, p = 0.004; Environment: F(3,59) = 2.2910, R2 = 0.10703, p = 0.001; Genetic Background*Environment: F(6,59) = 0.8725, R2 = 0.08153, p = 0.809. n = 15 bees/cage (social environment), 4 cages. Depicted as Principal Components Analysis (PCA) plots. Lowercase letters in legends denote statistically significant groups via pairwise comparisons. (D-H) Inoculated bees from different genetic backgrounds (D-F) and raised in different cage environments (D,G-H) differed in the relative abundance (D-E, G) of four individual microbial taxa and in the absolute abundance (F, H) of three individual microbial taxa. (D) All taxa depicted as stacked bar plots, with each bar representing a single bee’s gut microbial community. Asterisks in legend: *, p < 0.05 ANCOM-BC between genetic backgrounds; + , p < 0.05, ANCOM-BC between social environments. See Table 1 for all p values. (E-H) Significant taxa depicted as boxplots, lowercase letters represent statistically significant groups within each microbe via ANCOM-BC (relative abundance) or Permutation ANOVA (absolute abundance). See Tables 1 and 2 for all p values.
Table 3.
Relative abundance of each microbe in the gut microbial communities of control bees in each cage environment and genetic background (colony).
Fig 3.
Control bees remained microbiota-depleted, and showed similar differences in gut microbial community structure as treatment bees.
(A) Control (non-inoculated) bee gut microbiota were mostly composed of non-honey bee gut-associated bacteria, and differed in the abundance of some of these microbes based upon genetic background or cage environment. Depicted as stacked bar plots, with each bar representing a single bee’s gut microbial community. “Other” represents all non-honey bee gut-associated microbes that made up less than 1% of all samples. Asterisks in legend: *, p < 0.05 ANCOM-BC between genetic backgrounds; + , p < 0.05, ANCOM-BC between social environments. See Table 3 for all p values. (B) Control bees from different genetic backgrounds and in different cage environments marginally differed in gut microbiota alpha diversity. Shannon Alpha Diversity: Two-way ANOVA, Genetic Background: F(2,24) = 2.851, p = 0.078, Environment: F(2,24) = 3.562, p = 0.071, Genetic Background*Environment: F(2, 24) = 7.633, p = 0.003. (C) Control bees from different genetic backgrounds differed in gut microbiota beta diversity. Two-way Permutation MANOVA using Aitchison Distance, Genetic Background: F(2,29) = 1.69, R2 = 0.1063, p = 0.001; Environment: F(1,29) = 1.58, R2 = 0.0495, p = 0.002; Genetic Background*Environment: F(2,29) = 1.428, R2 = 0.0898, p = 0.003. n = 10 bees/colony (genetic background), 3 colonies. (D) Control bees raised in different cage environments differed in gut microbiota beta diversity. Two-way Permutation MANOVA using Aitchison Distance, Genetic Background: F(2,29) = 1.69, R2 = 0.1063, p = 0.001; Environment: F(1,29) = 1.58, R2 = 0.0495, p = 0.002; Genetic Background*Environment: F(2,29) = 1.428, R2 = 0.0898, p = 0.003. n = 15 bees/cage (social environment), 2 cages. Depicted as Principal Components Analysis (PCA) plots. Lowercase letters in legends denote statistically significant groups via pairwise comparisons. (E) Control bees from different cage environments differed in the relative abundance of six individual microbial taxa. Significant taxa depicted as boxplots, asterisks represent statistically significant differences within each microbe via ANCOM-BC. See Table 3 for all p values.
Table 4.
Absolute abundance of each microbe in the gut microbial communities of control bees in each cage environment and genetic background (colony).