Ultrastructure of the Bacteriocytes in the Midgut of the Carpenter ant Camponotus rufipes: Endosymbiont Control by Autophagy
It is known that the carpenter ant Camponotus rufipes has intracellular bacteria in bacteriocytes distributed in the midgut epithelium, each of which has varying quantities of endosymbionts depending on developmental stages. However, there is no comprehensive information on the midgut cells in adult workers. There are different kind of cells such as digestive cells, bacteriocytes, and cells with intermediate morphology make up the midgut epithelium. Autophagosomes and autolysosomes abound in the digestive and intermediate cells, all of which contain bacteria debris in the lümen which suggested that that the autophagy pathway regulates the endosymbiont level in C. rufipes midgut cells. Studies carried out to date have been shown that ın the midgut of carpenter ants, several genes associated with autophagy and lysosomes have been found, indicating a potential lysosomal pathway in the regulation of endosymbiont populations. These findings indicate some endosymbiont dynamics in Camponotus spp. midguts, but little is known about the organization of the midgut epithelium and the cellular events that control endosymbiont abundance. The aim of this study was to look at the morphology and cellular events that control the abundance of endosymbionts in C. rufipes workers’midgut cells in order to fill in these critical gaps and lead to a better understanding of the ant’s symbiotic relationships.
For these purposes; adult females of C. rufipes ants were collected and dissected to get midguts. Light Microscopy was used for histochemical studies. Fluorescence Microscopy was used to identify the presence of nucleic acid, midgut sections were obtained as a forementioned for light microscopy, except staining, and were incubated with DAPI nuclear stain. Samples were also analysed under Transmission Electron Microscopy and Confocal Microscopy for immunofluorescence analysis.
- Three cell types were found in the midgut:
- digestive cells with cytoplasm showing basophilic granules, amorphous vacuoles reaching to the midgut lumen with a well-developed striated border,
- bacteriocytes with acidophilic cytoplasm not reaching to the midgut lumen,
- few regenerative cells scattered in the basal region of the epithelium
The digestive cells were characterized by apical protrusions to the midgut lumen, rich in basophilic granules, which were positive for glycoconjugates and proteins. These cells had also amorphous vacuoles with some basophilic granules which were positive for proteins.
DAPI-stained slices showed that the cytoplasm of the midgut cells had different fluorescence signals. Cells with the weak fluorescence signals showed many granules and amorphous vacuoles with DAPI-positive content in the apical region.
The ultrastructure analysis of the midgut epithelium showed digestive cells closely associated with bacteriocytes.
Bacteriocytes presented an apical surface that did not reach the midgut lumen and had no microvilli, and the cytoplasm was filled with double-membrane bacteria with few mitochondria. The nucleus had a predominance of decondensed chromatin and well-developed nucleolus. The basal surface of these cells showed plasma membrane infoldings, forming a basal labyrinth with short extracellular spaces.
In the digestive cells, the apical surface presented well developed microvilli, cytoplasm rich in mitochondria, large amorphous compartments with electron-lucent content, electron-lucent vesicles, and lysosomes. In the basal cell region, the plasma membrane infoldings were long and formed a well-developed basal labyrinth of enlarged extracellular spaces associated with mitochondria.
There were cells with different ultrastructure, characterized by apical microvilli, cytoplasm-rich mitochondria, and electron-lucent vesicles, such as digestive cells, but they had also many bacteria, such as bacteriocytes. These cells will, henceforth, termed as intermediate cells.
- Both the digestive and intermediate cells had an apical cytoplasm with electron-lucent vesicles, lysosomes, autophagosome, and residual bodies rich in membranous content. In both these cell types, the apical cytoplasm had abundant bacteria inside vesicles closely associated with autophagosomes. These vesicles with bacteria were found fused with autophagosomes. To verify if the apical vesicles were autophagosomes, immunofluorescence detection to LC3A/B proteins revealed that they were undergoing autophagy.
In conclusion, the analysis indicates that autophagy controls the amount of endosymbionts in C. rufipes midgut cells. As a result, these cells transform from bacteriocytes that store endosymbionts to digestive cells. Some gaps in the evidence of endosymbionts in various midgut cell types, as well as bacteriocyte decrease during ant growth, can be better understood using this method. With their proposed model, some gaps in the evidence of endosymbionts in different midgut cell types and bacteriocyte decrease during ant growth can be better understood, potentially contributing to a better understanding of symbiotic relationships in carpenter ants.
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