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Vertical transmission of a Drosophila endosymbiont via cooption of the yolk transport and internalization machinery.

Herren JK, Paredes JC, Schüpfer F, Lemaitre B - MBio (2013)

Bottom Line: The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved.In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development.The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Science, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT

Unlabelled: Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity.

Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

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Spiroplasma colonization of the germ line. (A) Drawing showing the structure of an egg chamber, ovarioles. and ovary of Drosophila. The ovariole is surrounded by a muscular epithelium (light blue). Development of egg chambers progresses along the length of the ovariole, beginning with stem cell division in the germarium, which eventually becomes vitellogenic (accumulates yolk) and is termed the vitellarium, after which dumping of nurse cell contents into the oocyte occurs. Vitellogenic stage 10 egg chambers (left) are characterized by an outer layer of columnar follicle cells (green), an expanded oocyte (yellow), and nurse cells (blue). (B) Exterior surface of a vitellogenic stage 10 egg chamber. Spiroplasma bacteria are stained red (immunostaining with anti-Spiroplasma antibody), and cortical actin is stained green (with phalloidin), and this is the case for all subsequent images. (C) Spiroplasma localization in a series of egg chambers that represents the progression from germarium to vitellogenic oocytes. Germarium, stage 2, and stage 4, C1; stage 6, C2; stage 9, C3; stage 10, C4. (D) Actin staining of cell boundaries (D1) and Spiroplasma bacteria (D2) are merged (D3, D4) to show that Spiroplasma passes between follicle cells. D1 to D3 are transverse sections, and D4 is a longitudinal section. (E1) Bright-field image overlaid with fluorescent Spiroplasma staining reveals the localization of Spiroplasma bacteria in relation to yolk granules. In all of the fluorescence microscopy images, the scale bar represents 25 µm. (E2, E3) Electron micrograph showing localization of Spiroplasma bacteria (arrow) in relation to yolk granules (Y). In E1, Spiroplasma bacteria are contained between the yolk granule and the vesicular membrane. In E2, Spiroplasma bacteria can be seen partially penetrating the vesicular membrane. Spiroplasma cells could be identified in yolk granules on the basis of their size and morphology and the absence of such cells in yolk granules of flies without Spiroplasma bacteria.
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fig1: Spiroplasma colonization of the germ line. (A) Drawing showing the structure of an egg chamber, ovarioles. and ovary of Drosophila. The ovariole is surrounded by a muscular epithelium (light blue). Development of egg chambers progresses along the length of the ovariole, beginning with stem cell division in the germarium, which eventually becomes vitellogenic (accumulates yolk) and is termed the vitellarium, after which dumping of nurse cell contents into the oocyte occurs. Vitellogenic stage 10 egg chambers (left) are characterized by an outer layer of columnar follicle cells (green), an expanded oocyte (yellow), and nurse cells (blue). (B) Exterior surface of a vitellogenic stage 10 egg chamber. Spiroplasma bacteria are stained red (immunostaining with anti-Spiroplasma antibody), and cortical actin is stained green (with phalloidin), and this is the case for all subsequent images. (C) Spiroplasma localization in a series of egg chambers that represents the progression from germarium to vitellogenic oocytes. Germarium, stage 2, and stage 4, C1; stage 6, C2; stage 9, C3; stage 10, C4. (D) Actin staining of cell boundaries (D1) and Spiroplasma bacteria (D2) are merged (D3, D4) to show that Spiroplasma passes between follicle cells. D1 to D3 are transverse sections, and D4 is a longitudinal section. (E1) Bright-field image overlaid with fluorescent Spiroplasma staining reveals the localization of Spiroplasma bacteria in relation to yolk granules. In all of the fluorescence microscopy images, the scale bar represents 25 µm. (E2, E3) Electron micrograph showing localization of Spiroplasma bacteria (arrow) in relation to yolk granules (Y). In E1, Spiroplasma bacteria are contained between the yolk granule and the vesicular membrane. In E2, Spiroplasma bacteria can be seen partially penetrating the vesicular membrane. Spiroplasma cells could be identified in yolk granules on the basis of their size and morphology and the absence of such cells in yolk granules of flies without Spiroplasma bacteria.

Mentions: The Drosophila ovary consists of 15 to 18 discrete tubular ovarioles (Fig. 1A). Increasingly more mature egg chambers extend from the anterior to the posterior of the ovariole. The germ line stem cells are located at the anterior of the ovariole, in a region termed the germarium. Egg chamber development continues into the vitellarium region, where the oocyte takes up yolk and completes development into a fully formed unfertilized egg (14). Using immunofluorescence microscopy, we observed that Spiroplasma bacteria accumulate in the muscular epithelium that surrounds ovarioles. Specifically, Spiroplasma bacteria accumulate in the region of this epithelium that is proximal to the posterior end of the egg chamber (Fig. 1B), where endocytic activity is highest (15). We observed that Spiroplasma bacteria are not present in the germ line at the germarium stage, early in oogenesis (Fig. 1C1 and C2). Spiroplasma bacteria are known to be found occupying the abdominal cavity of females (16, 17). Therefore, to achieve vertical transmission, these bacteria must be able to reach the germ line at later oogenic stages from the hemolymph. We observed that Spiroplasma enters the germ line over specific stages of oogenesis (Fig. 1C3 and C4). Spiroplasma bacteria distinctly colonize the germ line during the vitellogenic stages (stages 8 to 10) of oogenesis, when yolk is incorporated into the oocyte. We also show that Spiroplasma bacteria are present in the extracellular space between follicle cells (Fig. 1D). In the oocyte, Spiroplasma bacteria are localized to vesicles like those formed by the endocytosis of yolk, also known as yolk granules (Fig. 1E1). Transmission electron microscopy (TEM) images reveal that Spiroplasma bacteria are found in the space between the yolk granule and the surrounding vesicular membrane (Fig. 1E2), which is consistent with a previous electron microscopy study (18). In our TEM images, we also observed Spiroplasma bacteria penetrating the vesicular membrane surrounding yolk granules (Fig 1E3); presumably, these cells are exiting yolk granules and gaining access to the oocyte cytoplasm. Altogether, this pattern of infection indicates that the route taken by Spiroplasma bacteria to reach the germ line involves invasion of the ovary, followed by passage between follicle cells of vitellogenic egg chambers, translocation across the oocyte membrane into the vesicles that become yolk granules, and finally traversal of the yolk vesicular membrane to access the oocyte cytoplasm.


Vertical transmission of a Drosophila endosymbiont via cooption of the yolk transport and internalization machinery.

Herren JK, Paredes JC, Schüpfer F, Lemaitre B - MBio (2013)

Spiroplasma colonization of the germ line. (A) Drawing showing the structure of an egg chamber, ovarioles. and ovary of Drosophila. The ovariole is surrounded by a muscular epithelium (light blue). Development of egg chambers progresses along the length of the ovariole, beginning with stem cell division in the germarium, which eventually becomes vitellogenic (accumulates yolk) and is termed the vitellarium, after which dumping of nurse cell contents into the oocyte occurs. Vitellogenic stage 10 egg chambers (left) are characterized by an outer layer of columnar follicle cells (green), an expanded oocyte (yellow), and nurse cells (blue). (B) Exterior surface of a vitellogenic stage 10 egg chamber. Spiroplasma bacteria are stained red (immunostaining with anti-Spiroplasma antibody), and cortical actin is stained green (with phalloidin), and this is the case for all subsequent images. (C) Spiroplasma localization in a series of egg chambers that represents the progression from germarium to vitellogenic oocytes. Germarium, stage 2, and stage 4, C1; stage 6, C2; stage 9, C3; stage 10, C4. (D) Actin staining of cell boundaries (D1) and Spiroplasma bacteria (D2) are merged (D3, D4) to show that Spiroplasma passes between follicle cells. D1 to D3 are transverse sections, and D4 is a longitudinal section. (E1) Bright-field image overlaid with fluorescent Spiroplasma staining reveals the localization of Spiroplasma bacteria in relation to yolk granules. In all of the fluorescence microscopy images, the scale bar represents 25 µm. (E2, E3) Electron micrograph showing localization of Spiroplasma bacteria (arrow) in relation to yolk granules (Y). In E1, Spiroplasma bacteria are contained between the yolk granule and the vesicular membrane. In E2, Spiroplasma bacteria can be seen partially penetrating the vesicular membrane. Spiroplasma cells could be identified in yolk granules on the basis of their size and morphology and the absence of such cells in yolk granules of flies without Spiroplasma bacteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Spiroplasma colonization of the germ line. (A) Drawing showing the structure of an egg chamber, ovarioles. and ovary of Drosophila. The ovariole is surrounded by a muscular epithelium (light blue). Development of egg chambers progresses along the length of the ovariole, beginning with stem cell division in the germarium, which eventually becomes vitellogenic (accumulates yolk) and is termed the vitellarium, after which dumping of nurse cell contents into the oocyte occurs. Vitellogenic stage 10 egg chambers (left) are characterized by an outer layer of columnar follicle cells (green), an expanded oocyte (yellow), and nurse cells (blue). (B) Exterior surface of a vitellogenic stage 10 egg chamber. Spiroplasma bacteria are stained red (immunostaining with anti-Spiroplasma antibody), and cortical actin is stained green (with phalloidin), and this is the case for all subsequent images. (C) Spiroplasma localization in a series of egg chambers that represents the progression from germarium to vitellogenic oocytes. Germarium, stage 2, and stage 4, C1; stage 6, C2; stage 9, C3; stage 10, C4. (D) Actin staining of cell boundaries (D1) and Spiroplasma bacteria (D2) are merged (D3, D4) to show that Spiroplasma passes between follicle cells. D1 to D3 are transverse sections, and D4 is a longitudinal section. (E1) Bright-field image overlaid with fluorescent Spiroplasma staining reveals the localization of Spiroplasma bacteria in relation to yolk granules. In all of the fluorescence microscopy images, the scale bar represents 25 µm. (E2, E3) Electron micrograph showing localization of Spiroplasma bacteria (arrow) in relation to yolk granules (Y). In E1, Spiroplasma bacteria are contained between the yolk granule and the vesicular membrane. In E2, Spiroplasma bacteria can be seen partially penetrating the vesicular membrane. Spiroplasma cells could be identified in yolk granules on the basis of their size and morphology and the absence of such cells in yolk granules of flies without Spiroplasma bacteria.
Mentions: The Drosophila ovary consists of 15 to 18 discrete tubular ovarioles (Fig. 1A). Increasingly more mature egg chambers extend from the anterior to the posterior of the ovariole. The germ line stem cells are located at the anterior of the ovariole, in a region termed the germarium. Egg chamber development continues into the vitellarium region, where the oocyte takes up yolk and completes development into a fully formed unfertilized egg (14). Using immunofluorescence microscopy, we observed that Spiroplasma bacteria accumulate in the muscular epithelium that surrounds ovarioles. Specifically, Spiroplasma bacteria accumulate in the region of this epithelium that is proximal to the posterior end of the egg chamber (Fig. 1B), where endocytic activity is highest (15). We observed that Spiroplasma bacteria are not present in the germ line at the germarium stage, early in oogenesis (Fig. 1C1 and C2). Spiroplasma bacteria are known to be found occupying the abdominal cavity of females (16, 17). Therefore, to achieve vertical transmission, these bacteria must be able to reach the germ line at later oogenic stages from the hemolymph. We observed that Spiroplasma enters the germ line over specific stages of oogenesis (Fig. 1C3 and C4). Spiroplasma bacteria distinctly colonize the germ line during the vitellogenic stages (stages 8 to 10) of oogenesis, when yolk is incorporated into the oocyte. We also show that Spiroplasma bacteria are present in the extracellular space between follicle cells (Fig. 1D). In the oocyte, Spiroplasma bacteria are localized to vesicles like those formed by the endocytosis of yolk, also known as yolk granules (Fig. 1E1). Transmission electron microscopy (TEM) images reveal that Spiroplasma bacteria are found in the space between the yolk granule and the surrounding vesicular membrane (Fig. 1E2), which is consistent with a previous electron microscopy study (18). In our TEM images, we also observed Spiroplasma bacteria penetrating the vesicular membrane surrounding yolk granules (Fig 1E3); presumably, these cells are exiting yolk granules and gaining access to the oocyte cytoplasm. Altogether, this pattern of infection indicates that the route taken by Spiroplasma bacteria to reach the germ line involves invasion of the ovary, followed by passage between follicle cells of vitellogenic egg chambers, translocation across the oocyte membrane into the vesicles that become yolk granules, and finally traversal of the yolk vesicular membrane to access the oocyte cytoplasm.

Bottom Line: The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved.In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development.The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Science, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT

Unlabelled: Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity.

Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

Show MeSH
Related in: MedlinePlus