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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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Involvement of Yolkless receptor-mediated endocytosis in Spiroplasma transmission. (A) Spiroplasma levels in flies and embryos are shown for the control (ORR) and yolkless (yl13) mutants. Spiroplasma levels were monitored by qPCR with a Spiroplasma-specific gene (dnaA). Each value was normalized to the average of the control values for that experiment (ORR flies or embryos), which was set at 100%. All of the repeats from all of the experiments were then pooled. The number of samples collected independently for DNA extraction is shown by the value in each bar. Error bars represent the standard error of the mean. NS and *** denote levels of statistical significance in a Mann-Whitney U test of difference when yl13 mutants are compared to the control (ORR) for flies (P = 0.6298) and for embryos laid by these flies (P < 0.0001). (B) Stage 10 oocytes from control (ORR) (B1), yl13 (B2), and Rab52-deficient germ line clone (B3) flies. Note that Rab52-deficient clones exhibit structural deformations. The arrowhead in B2 denotes an accumulation of Spiroplasma bacteria between the follicle cells surrounding yl13 mutant oocytes. The arrowhead in B3 denotes actin-rich cortical invagination that is associated with the presence of Spiroplasma (see image analysis in Fig. S1 in the supplemental material). The images at the bottom are higher magnifications of the insets.
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fig2: Involvement of Yolkless receptor-mediated endocytosis in Spiroplasma transmission. (A) Spiroplasma levels in flies and embryos are shown for the control (ORR) and yolkless (yl13) mutants. Spiroplasma levels were monitored by qPCR with a Spiroplasma-specific gene (dnaA). Each value was normalized to the average of the control values for that experiment (ORR flies or embryos), which was set at 100%. All of the repeats from all of the experiments were then pooled. The number of samples collected independently for DNA extraction is shown by the value in each bar. Error bars represent the standard error of the mean. NS and *** denote levels of statistical significance in a Mann-Whitney U test of difference when yl13 mutants are compared to the control (ORR) for flies (P = 0.6298) and for embryos laid by these flies (P < 0.0001). (B) Stage 10 oocytes from control (ORR) (B1), yl13 (B2), and Rab52-deficient germ line clone (B3) flies. Note that Rab52-deficient clones exhibit structural deformations. The arrowhead in B2 denotes an accumulation of Spiroplasma bacteria between the follicle cells surrounding yl13 mutant oocytes. The arrowhead in B3 denotes actin-rich cortical invagination that is associated with the presence of Spiroplasma (see image analysis in Fig. S1 in the supplemental material). The images at the bottom are higher magnifications of the insets.

Mentions: To test this hypothesis, we used quantitative PCR (qPCR) and fluorescence microscopy to quantify Spiroplasma transmission in flies lacking the yolk receptor, Yolkless. yl13 is a strong loss-of-function mutation in yolkless that causes a marked decrease in yolk uptake by oocytes and an increase in the amount of yolk in the hemolymph (21). Without sufficient yolk, the eggs laid by yl13 mutant flies do not complete early embryonic development (21). We first quantified Spiroplasma titers by qPCR of the Spiroplasma dnaA gene in control Oregon-R (ORR) and yl13 homozygous female flies, as well as in their embryos. Although we initially quantified the Spiroplasma dnaA copy number relative to that of a host nuclear gene, RPS17, we decided to remove this from all of our analyses because the host nuclear gene copy number was, unsurprisingly, much lower in nonviable eggs. We observed that yl13 homozygous flies transmit four times fewer Spiroplasma bacteria to eggs than their wild-type counterparts do, while Spiroplasma levels in whole flies are not significantly different (Fig. 2A). Consistent with these observations, immunofluorescence microscopy revealed that oocytes of yolkless homozygous infected females contained much lower Spiroplasma levels than did wild-type oocytes of the same stage (compare Fig. 2B1 and B2; see Fig. S1A in the supplemental material for image quantification). We observed that, in some cases, Spiroplasma bacteria tended to accumulate between follicle cells and on the outer surface of the yl13 oocyte, suggesting that the blockage occurs at the point of oocyte entry. We observed a similar phenotype when using another independently derived mutant allele of yolkless, yl15.


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)

Involvement of Yolkless receptor-mediated endocytosis in Spiroplasma transmission. (A) Spiroplasma levels in flies and embryos are shown for the control (ORR) and yolkless (yl13) mutants. Spiroplasma levels were monitored by qPCR with a Spiroplasma-specific gene (dnaA). Each value was normalized to the average of the control values for that experiment (ORR flies or embryos), which was set at 100%. All of the repeats from all of the experiments were then pooled. The number of samples collected independently for DNA extraction is shown by the value in each bar. Error bars represent the standard error of the mean. NS and *** denote levels of statistical significance in a Mann-Whitney U test of difference when yl13 mutants are compared to the control (ORR) for flies (P = 0.6298) and for embryos laid by these flies (P < 0.0001). (B) Stage 10 oocytes from control (ORR) (B1), yl13 (B2), and Rab52-deficient germ line clone (B3) flies. Note that Rab52-deficient clones exhibit structural deformations. The arrowhead in B2 denotes an accumulation of Spiroplasma bacteria between the follicle cells surrounding yl13 mutant oocytes. The arrowhead in B3 denotes actin-rich cortical invagination that is associated with the presence of Spiroplasma (see image analysis in Fig. S1 in the supplemental material). The images at the bottom are higher magnifications of the insets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Involvement of Yolkless receptor-mediated endocytosis in Spiroplasma transmission. (A) Spiroplasma levels in flies and embryos are shown for the control (ORR) and yolkless (yl13) mutants. Spiroplasma levels were monitored by qPCR with a Spiroplasma-specific gene (dnaA). Each value was normalized to the average of the control values for that experiment (ORR flies or embryos), which was set at 100%. All of the repeats from all of the experiments were then pooled. The number of samples collected independently for DNA extraction is shown by the value in each bar. Error bars represent the standard error of the mean. NS and *** denote levels of statistical significance in a Mann-Whitney U test of difference when yl13 mutants are compared to the control (ORR) for flies (P = 0.6298) and for embryos laid by these flies (P < 0.0001). (B) Stage 10 oocytes from control (ORR) (B1), yl13 (B2), and Rab52-deficient germ line clone (B3) flies. Note that Rab52-deficient clones exhibit structural deformations. The arrowhead in B2 denotes an accumulation of Spiroplasma bacteria between the follicle cells surrounding yl13 mutant oocytes. The arrowhead in B3 denotes actin-rich cortical invagination that is associated with the presence of Spiroplasma (see image analysis in Fig. S1 in the supplemental material). The images at the bottom are higher magnifications of the insets.
Mentions: To test this hypothesis, we used quantitative PCR (qPCR) and fluorescence microscopy to quantify Spiroplasma transmission in flies lacking the yolk receptor, Yolkless. yl13 is a strong loss-of-function mutation in yolkless that causes a marked decrease in yolk uptake by oocytes and an increase in the amount of yolk in the hemolymph (21). Without sufficient yolk, the eggs laid by yl13 mutant flies do not complete early embryonic development (21). We first quantified Spiroplasma titers by qPCR of the Spiroplasma dnaA gene in control Oregon-R (ORR) and yl13 homozygous female flies, as well as in their embryos. Although we initially quantified the Spiroplasma dnaA copy number relative to that of a host nuclear gene, RPS17, we decided to remove this from all of our analyses because the host nuclear gene copy number was, unsurprisingly, much lower in nonviable eggs. We observed that yl13 homozygous flies transmit four times fewer Spiroplasma bacteria to eggs than their wild-type counterparts do, while Spiroplasma levels in whole flies are not significantly different (Fig. 2A). Consistent with these observations, immunofluorescence microscopy revealed that oocytes of yolkless homozygous infected females contained much lower Spiroplasma levels than did wild-type oocytes of the same stage (compare Fig. 2B1 and B2; see Fig. S1A in the supplemental material for image quantification). We observed that, in some cases, Spiroplasma bacteria tended to accumulate between follicle cells and on the outer surface of the yl13 oocyte, suggesting that the blockage occurs at the point of oocyte entry. We observed a similar phenotype when using another independently derived mutant allele of yolkless, yl15.

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