Figure 1.
Adults fly into the dung pat to feed and mate. Beneath the dung pat, the juvenile life stages of Onthophagus taurus are isolated in the brood chamber of the brood balls constructed by the female beetles in tunnels. Females lay several brood balls in each tunnel that would all be at the same developmental stage. However, for illustrative purposes all life stages are represented in one tunnel. These stages include the: (A) egg, (B) 1st larval instar, (C) 2nd larval instar, (D) 3rd larval instar, (E) pupa, and (F) an eclosing adult beetle that is tunneling toward the surface. The brood ball chamber is larger with each successive life stage as the larva feeds on the chamber walls within the brood ball. The top inset shows (G) the fecal pedestal the egg is positioned upon in brood ball. The bottom inset shows (H) the larval instar feeding on the walls of the brood ball chamber.
Figure 2.
The brood ball chamber is a unique structure for the transmitted microbiome of dung beetles. (A) The innermost walls of the brood ball chamber with the egg are smooth as compared to the fibrous outer walls. (B) A brood ball is shown broken into 3 pieces. The hollow brood ball chamber is where the egg develops (left). The remainder of the brood ball is filled with cellulose-rich, fibrous dung (center and right). Images C-F are scanning electron micrographs of different portions of the brood ball. (C) Micrograph illustrating the smooth biofilm-like matrix coating the inner wall of the brood ball chamber. (D) Fibrous bits of grass from the cow dung found in the portion of the brood ball away from the chamber. (E) Higher magnification of the smooth biofilm-like matrix that coats the brood ball chamber walls. (F) When the smooth matrix is scraped away, rod shaped microbes in chains (arrows) are found underneath the smooth matrix of the brood ball chamber walls. Other rod-shaped structures in the background remain covered in the biofilm-like matrix (arrowheads). This matrix and microbes are only observed where the mother has prepared the brood ball chamber.
Figure 3.
Relative abundance of family level OTUs composing the dung beetle microbiome.
The relative abundance of each taxon is shown for each sample. Enterobacteriaceae (bright blue) were found in 95% of the samples, in all populations, and all life stages. Other major components include bacteria in the Comamonadaceae (green) and Pseudomonadaceae (bluish purple). Sample names are abbreviated as follows: 1L (1st instar larva), 2L (2nd instar larva), 3L (3rd instar larva), PU (pupa), CI (cultured isolates), FP (female parent), IN (Indiana population), and NC (North Carolina population). The number after the life stage indicates different individuals sampled.
Figure 4.
Clustering of samples based on microbiome composition.
The relative proportion of sequences in a specific OTU (color key in upper right corner) is illustrated on a heat map with samples clustered using a furthest neighbor clustering to determine similarity of both the composition and abundance of the microbiomes. Several OTUs were only seen once. Overall, samples did not cluster by population, parental female, life stage, or brood ball age except the cultured isolates (CI3NC1 and CI4NC1) from the digestive system of the 3rd instar larvae. Interestingly, the four cultured samples were oviposited at the same time; however, samples CI3NC1 and CI4NC1 that cluster were sampled 7 d later than CI1NC1 and CI2NC1. Thus, samples CI3NC1 and CI4NC1 were older and were transitioning from the 3rd larval instar to the pupal stages. Each sample is labeled by the source (CI=cultured isolate, 1L=1st instar, 3L=3rd instar, 2L=2nd instar, Pu=pupae, FP= female parent), the isolate or individual animal number, the US state from which the parental female was isolated (NC =North Carolina populations, USA, and IN=Indiana, USA), and the female parent number (NC1, NC2, IN1).
Figure 5.
Abundance of lowest common ancestors across all microbiome samples.
Bacterial OTUs identified in Qiime are arranged in a bulls-eye configuration with the most commonly occurring group in the yellow center, those occurring in two or more individuals in the blue circle, and those genera occurring only one time in the outermost rim. The lowest common ancestor Qiime classification is given and may be at the genus, family, or class level and as such may overlap. Enterobacter was found in offspring of all parental females and in 68% of the individuals. Samples denoted with a star are those OTUs that were found using both culture-dependent and -independent techniques.
Figure 6.
An unrooted phylogenomic tree based on 287 loci from 1095 sequenced bacterial genomes was made as a skeletal tree on which was placed the lepA and gyrB sequences from 16 bacterial cultures isolated from the digestive system of four different 3rd instar larvae from female parent 2 from the North Carolina population. This analysis placed the isolates sister to (A) Providencia stuartii, (B) Enterobacter cloacae, (C) Pusillimonas, (D) Pedobacter heparinus, and (E) Lysinibacillus sphaericus.