Male killing Spiroplasma preferentially disrupts neural development in the Drosophila melanogaster embryo

Abstract

Male killing bacteria such as Spiroplasma are widespread pathogens of numerous arthropods including Drosophila melanogaster. These maternally transmitted bacteria can bias host sex ratios toward the female sex in order to 'selfishly' enhance bacterial transmission. However, little is known about the specific means by which these pathogens disrupt host development in order to kill males. Here we show that a male-killing Spiroplasma strain severely disrupts nervous tissue development in male but not female D. melanogaster embryos. The neuroblasts, or neuron progenitors, form properly and their daughter cells differentiate into neurons of the ventral nerve chord. However, the neurons fail to pack together properly and they produce highly abnormal axons. In contrast, non-neural tissue, such as mesoderm, and body segmentation appear normal during this time, although the entire male embryo becomes highly abnormal during later stages. Finally, we found that Spiroplasma is altogether absent from the neural tissue but localizes within the gut and the epithelium immediately surrounding the neural tissue, suggesting that the bacterium secretes a toxin that affects neural tissue development across tissue boundaries. Together these findings demonstrate the unique ability of this insect pathogen to preferentially affect development of a specific embryonic tissue to induce male killing.

Figure 1. Spiroplasma alters general morphology of the whole male during late embryogenesis.

All embryos shown are MSRO-infected. Insets depict DNA (blue) and a Y-chromosome specific probe (red), which indicates sex of the embryo.

Figure 2. Spiroplasma-infected male embryos exhibit normal neuroblasts but highly irregular neurons.

All embryos shown are MSRO-infected. The neuroblasts are highlighted by anti-Deadpan (red in top left panels); the neuron cell bodies are marked by anti-Elav (green in bottom left panels); the neuron axons are marked by the 22C10 antibody (green in top right panels); and neurons, glial cells, and axons are indicated by anti-Neuroglian (green in bottom right panels).

Figure 3. Spiroplasma does not cause abnormal levels of cell proliferation, cell death, or abnormal defects in non-neural tissues or other processes during the onset of neural defects.

All embryos shown are MSRO-infected. Cell proliferation is depicted by anti-phospho histone H3 (PH3) (red in top left panels); broken DNA, an indicator of cell death, is shown by TUNEL (red in middle left panels); The ventral midline invagination can be seen with DNA staining (indicated by white arrows in bottom left panels); The developing mesoderm is highlighted by anti-Twist (red in top right panels); A subset of motoneurons located in odd body segments is marked by anti-even-skipped (white in middle right panels); external body segments are seen through DNA staining (indicated by white arrows in bottom right panels).

Figure 4. Spiroplasma is completely absent from the neural tissue but heavily concentrates in other tissues of both male and female embryos.

High levels of bacteria (red) are located in the yolky interior of the cleavage blastoderm, while few bacteria can be seen around the cells of the epithelium (top panels). During later embryonic stages, no bacteria can be seen to overlap with the cells of the ventral nerve chord (insets in the bottom panels). During this time high levels of Spiroplasma are present both inside and outside the gut (bottom panels).