Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus

Abstract

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.

FIG. 1.

Bacteriocytes in the midgut of a C. floridanus worker. (Left) Methylene blue-stained thin section of the midgut; (right) electron micrograph showing an enlargement of a typical region. Ent, enterocyte; Mv, microvilli; Bac, bacteriocyte; Nu, nucleus.

FIG. 2.

Bacteriocytes in the midgut of a virgin queen. (Left) methylene blue-stained thin section of the midgut epithelium; (right) electron micrograph showing an enlargement of a typical region. For abbreviations, see the legend to Fig. 1.

FIG. 3.

Occurrence of endosymbiotic bacteria in bacteriocytes and enterocytes of a male derived from a queen. The electron micrograph shows a typical bacteriocyte filled with bacteria and nearby enterocytes also harboring the endosymbionts, some of which are marked with white arrows. For abbreviations, see the legend to Fig. 1.

FIG. 4.

Empty bacteriocytes in the midgut of a mature queen. (Left) Methylene blue-stained thin section of the midgut epithelium; (right) electron micrograph showing an enlargement of a typical region. For abbreviations, see the legend to Fig. 1.

FIG. 5.

(A) Ovary of a mature queen showing an oocyte (Ooc) filled with bacteria and associated follicle cells (Fc). (Left) Methylene blue-stained thin section; (right) electron micrograph of an oocyte surrounded by follicle cells. (B) Ovary of a worker showing an oocyte (Ooc) filled with bacteria and associated follicle cells (Fc). (Left) methylene blue-stained thin section; (right) electron micrograph of the cytoplasm of an oocyte. For abbreviations, see the legend to Fig. 1.

FIG. 6.

(A) The spermatheca of a mature queen does not harbor endosymbiotic bacteria. (Left) Methylene blue-stained thin section of the spermatheca; (right) electron micrograph. (B) The testicle of a male harbors only few endosymbiotic bacteria. (Left) Methylene blue-stained thin section of the testicle; (right) electron micrograph. The arrows indicate the presence of some bacteria.

FIG. 7.

In situ hybridization with digoxigenin-labeled C. floridanus-specific oligonucleotides of cryosections of various developmental stages. A black area indicates the presence of bacteria. (A) Freshly laid egg; (B) 1-mm larva; (C) 3 mm larva; (D) 6-mm larva; (E) negative control.

FIG. 7.

In situ hybridization with digoxigenin-labeled C. floridanus-specific oligonucleotides of cryosections of various developmental stages. A black area indicates the presence of bacteria. (A) Freshly laid egg; (B) 1-mm larva; (C) 3 mm larva; (D) 6-mm larva; (E) negative control.

FIG. 8.

Electron micrograph of the midgut tissue of C. floridanus immediately after the end of tetracycline treatment. The arrows indicate the presence of several residual bacteria in this particular bacteriocyte. For abbreviations, see the legend to Fig. 1.