The male-killing Spiroplasmas of Drosophila nebulosa and Drosophila willistoni have identical ITS sequences.
Bentley, Joanna K., Georgina Hinds, and Gregory D.D. Hurst. Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London, NW1 4HE, UK. E-mail: email@example.com
Several species of the Drosophila willistoni group were first shown to carry male-killing bacteria during the 1950’s. Certain females collected from the field produced only daughters, their sons being killed during embryogenesis. This trait was shown to be maternally inherited and associated with a Spiroplasma bacterium that killed male hosts only (reviewed in Williamson and Poulson, 1979). Male-killing bacteria have since been found in many insect species, and several bacteria have been shown to exhibit male-killing (Hurst and Jiggins, 2001). The Spiroplasma of D. willistoni has been cultured and formally described as Spiroplasma poulsonii (Williamson et al., 1999).
The aim of this study was to investigate the closeness of the relationship between Spiroplasma strains from different Drosophila species. Do different species of fly have similar Spiroplasma, or are they different Spiroplasma altogether? If they are similar Spiroplasma, can we say whether they are similar because of shared ancestry or because of horizontal transmission of the bacterium between members of the group? Previous studies have shown all male-killing bacteria in these flies have similar morphology. However, the observation that mixing of infections from two different willistoni species leads to a ‘clumping’ reaction, in which all the Spiroplasma stick together, indicates they are not identical (Williamson and Poulson, 1979).
The relative ease of molecular phylogenetic analysis has made it possible for us to reassess the relationship between the different strains. The 16S-23S ribosomal spacer region (ITS; intergenic transcribed spacers) has been used in previous studies to investigate bacterial genetic relationships (Daffonchio et al., 1998; Jensen et al., 1993). Because it is a non-coding region it is more variable and therefore more informative in differentiating between close relatives than the traditional 16S rDNA sequence (Aakra et al., 1999). Schulenburg et al. (2000) used this region in their study of male-killing Spiroplasma relationships and concluded that the variability in this region of sequence would facilitate its use as a species-specific marker, evolving at double the rate of 16S rDNA.
The ITS region was sequenced for two recently collected D. nebulosa infections from Guadeloupe and also for a Spiroplasma infection originating from D. nebulosa and microinjected into D. melanogaster over forty years ago. Primers JO4 and N2 were used and the method in Schulenburg et al. (2000) followed, giving a sequence length of around 450bp. The new Spiroplasma sequences were all identical to that of S. poulsonii from D. willistoni (Schulenburg et al., 2000, Accession no. AJ130995).
We conclude, therefore, that these Spiroplasma strains are closely related. What is unsure is whether they are similar because of common descent (the bacterium was present and maintained in these flies since their split), or whether they are similar because the bacterium has transmitted horizontally between host species. We can approach this issue by comparing the divergence of host and Spiroplasma.
The sequence divergence between different bacteria can be calculated by reference to the 16S ‘clock’ gene (Moran et al., 1993) which has a lineage divergence of 1% per 50ma (2% sequence divergence). The ITS sequence evolves at double this rate, i.e., 1% per 25ma (2% sequence divergence) (Schulenburg et al., 2000). The date of the divergence between D. nebulosa and D. willistoni can be calculated by comparing COI sequences. Brower (1994) showed the rate of divergence of COI in Drosophila is 1.1% per million years per lineage at silent sites (2.2% sequence divergence). The COI sequences from D. nebulosa (U51605) and D. willistoni (U51589) (Gleason et al., 1998) were found to have 25 silent sites and 2 non silent sites over 471 bases. This gives an estimated divergence time around 7.2 million years ago (i.e., ca. 5-10 million years ago) for the separation of D. willistoni and D. nebulosa. Unfortunately this means we cannot tell if horizontal transmission has occurred. During this timeframe, we would expect the ITS sequence of the Spiroplasma, to have accumulated 2-3 mutations, using the ‘clock’ above. Whilst absence of divergence in the ITS sequence is consistent with horizontal transmission, the low level of divergence expected with common ancestry forbids us from making the conclusion that their relatedness is because of a recent horizontal transmission event.
In conclusion, the D. nebulosa Spiroplasma strains are closely related to that of S. poulsonii in D. willistoni, and we can consider this a single instance of male-killing. However, the clumping reaction observed previously indicates subtle genetic differentiation, and we cannot formally distinguish between common ancestry and horizontal transmission as the cause of their close relationship.
Acknowledgments: We would like to thank Dr. T. Koana for the stock of D. melanogaster infected with NSRO.
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