Spiroplasma poulsonii sp. nov., a new species associated with male-lethality in Drosophila willistoni, a neotropical species of fruit fly

Progenies from some wild-caught females of Drosophila willistoni and three other sibling species are entirely female. The proclivity for production of unisexual female progeny by these flies was named the sex ratio (SR) trait and was originally thought to be genetic. However, experiments in the laboratory of Donald F. Poulson in the early 1960s demonstrated that this ‘trait’ was vertically transmitted and infectious, in that it could be artificially transferrd by injection from infected females to non-infected females. Motile, helical micro-organisms were observed in females showing the trait. In 1979, the SR organisms were designated as group II in the informal spiroplasma classification system. The organisms proved to be extremely fastidious, but were eventually cultivated in a very complex cell-free medium (H-2) after initial co-cultivation with insect cells. Cultivation in the H-2 medium and the subsequent availability of a triply cloned strain (DW-1T) permitted comparaltiv studies. Cells of strain DW-1Twere helical, motile filaments 200--250 nm in diameter and were bound by a single trilaminar membrane. Cells plated on 1.8% Noble agar formed small satellite-free colonies 60--70 μm in diameter with dense centres and uneven edges. The temperature range for growth was 26--30 °C; optimum growth occurred at 30 °C, with a doubling time in H-2 medium of 15.8 h. The strain passed through filters with 220 nm, but not 100 nm, pores. Reciprocal serological comparisons of strain DW-1Twith representatives of other spiroplasma groups showed an extensive pattern of one-way crossing when strain DW-1Twas used as antigen. However, variable, usually low-level reciprocal cross-reactions were observed between strain DW 1Tand representatives of group I sub-groups. The genome size of strain DW-1Twas 2040 kbp, as determined by PFGE. The G+C content was 26·1 mol%, as determined by buoyant density and melting point methods. The serological and molecular data indicate that strain DW-1Tis separated from group I representative strains sufficiently to justify retention of its group status. Continued group designation is also indicated by the ability of SR spiroplasma to induce male lethality in Drosophila, their vertical transmissibility and their extremely fastidious growth requirements. Group II spiroplasmas, represented by strain DW-1T(ATCC 43153T), are designated Spiroplasma poulsonii.