Neural primordium as a target of Spiroplasma-induced male killing in Drosophila melanogaster. Trisha N Chong, Jennifer C Martin, Patrick M Ferree. W. M. Keck Science Department, The Claremont Colleges, Claremont, CA.

   Male killing bacteria are widespread pathogens of the arthropods and can have dramatic effects on the population dynamics of these host organisms. However, the specific molecular and cellular targets are currently unknown. We have performed developmental analyses of D. melanogaster males that are targeted by the male killing bacterium, Spiroplasma poulsonii. Previous studies in another host species, D. nebulosa, suggested that male killing occurs during two phases: embryonic and larval. We used sex-specific fluorescence probes and the yellow1 visible marker in order to distinguish between infected D. melanogaster males and females at all developmental stages so that we could carefully analyze the development of both sexes. We found that over 99% of infected male embryos fail to hatch into first instar larvae. Rare escaper males that developed to the third instar stage exhibited only marginally reduced body size, salivary gland size, brain size, and brain cell mitotic index. Thus, the primary lethal phase in this host-pathogen system is embryonic. Confocal microscopic analyses of dying male embryos revealed that early cleavage stages progressed normally. However, stage 12-13 embryos showed dramatic morphological defects that mapped to the neural primordium. Using an antibody against Neuroglian to visualize the neural tissues, we observed that the neural primordium became increasingly disorganized during later stages, with severely broken glanglia and irregular ventral nerve cord. In contrast, mesoderm and endoderm-derived tissues appeared normal during stage 12-13 and also at earlier stages. These results suggest that the male-killing effect originates specifically in the neural tissues. Additionally, our studies revealed that ~40% of females die as embryos. However, female embryos show normal neural morphology. Thus, Spiroplasma kills males specifically by targeting the neural tissues but also kills female embryos in an alternate manner. These findings will greatly facilitate ongoing studies aimed at identifying the exact molecular target(s) of male killing.