Table 1. Normalized average relative expression of spermatogenesis-related transcripts in whole bodies and testes alone. Tissue Transcript Whole bodies always early 1.210 cookie monster 5.086 Testes alone always early don juan Mst84D don juan Mst84D cookie monster 0.602 1.748 1.545 1.011 1.521 0.737 F1 hybrid D. simulans D. mauritiana males 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.154 0.029 0.371 0.116 0.801 0.888 0.610 0.310 doi:10.1371/journal.pone.0003009.t001 normalized on an arbitrary scale where the D. mauritiana average was set to 1.00 through simple division. Statistical analysis was performed with StatView (SAS Institute, Cary, NC). Samples were compared via ANOVA, but qualita- tively similar results were obtained when using nonparametric pairwise comparative statistics such as the Mann-Whitney U-test. Results Using our RNA preparations, we were able to obtain real-time PCR quantitations of reverse transcribed genes from both whole bodies and testes alone. Using RNA derived from whole bodies, we found that all four transcripts (aly, comr, dj, Mst84D) were all expressed in F1 hybrid males at levels significantly lower than in both parent species (see Table 1). One sample of D. simulans comr was anomalously higher than the rest (three times higher than the rest), skewing the figure presented; however, excluding this sample still yielded a significant difference. The degree of underexpression was substantially reduced when testes were examined alone (Table 1, Figure 2). For the genes aly and comr, there was extensive overlap in expression between the F1 hybrid samples and both pure-species samples, demonstrating the lack of a consistent difference in expression between hybrids and pure species at these early-acting loci. We did observe a significant difference in expression at Mst84D between pure-species and hybrids (F1 vs. D. simulans, p = 0.0039, F1 vs. D. mauritiana, p = 0.0007) when testes were examined alone. The difference in testes expression at dj was also significant between D. simulans and the F1 hybrids (p = 0.0022), but one extreme outlier sample of D. mauritiana (having expression one third that of all of the other samples) made the expression difference between these samples Drosophila Hybrid Disruptions and the F1 not statistically significant. However, if this extreme outlier (which also had much weaker PCR amplification of both the experimental and control genes) was excluded, this difference in expression was also significant (p = 0.0086). Discussion This study presents two conceptual advances and one technical advance for understanding the role of gene expression disruptions in Drosophila hybrid sterility. Studies of hybrid male misexpres- sion (over- or under-expression relative to both pure species) have either focused on whole bodies or on RNA isolated from testes. Here, we use both techniques on hybrids of Drosophila simulans and D. mauritiana, and we find that some transcripts are underexpressed when the whole body is studied but not underexpressed when testes are surveyed alone. This difference may partially explain the discrepancy in results from the studies of Haerty and Singh (89 underexpressed genes in testes: [11]) and Moehring et al. (502 underexpressed genes in whole bodies: [13]) when surveying hybrids of the same species pairs. Additionally, using assays of four genes, we found some support for the hypothesis of Moehring et al. [13] that genes late in the spermatogenesis pathway (Mst84D and don juan) are more likely to be underexpressed in hybrid testes than those early in the spermatogenesis pathway (always early and cookie monster, see Figure 1). While this conclusion is tentative because we cannot necessarily extrapolate from assays of only four loci, it nonetheless suggests the possibility that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes. Several hypotheses can explain the difference in detected expression between whole bodies and testes alone. Since the testes appear to be of comparable size and structure in all flies surveyed, some of the transcripts identified by Moehring et al. [13] may be underexpressed in other tissues. However, evidence for non-testes expression of these transcripts in other tissues is minimal. Very low levels of always early and cookie monster are suggested by microarray analysis of various tissues [24], but RT-PCR or northern blots of gonadectomized or germline-less adult males failed to detect them [18,25]. Alternatively, there may be developmental (or very broad regulatory) disruptions present in testes of these sterile hybrids that are not apparent with microscopic visualization. For example, if testes of sterile hybrids have reduced testes transcription overall (or even just reduced expression of the control gene), then testes- specific transcripts would be detected as underexpressed in RNA preparations from whole bodies but not necessarily in RNA preparations from testes alone. Consistent with this hypothesis, donjuan and Mst84D appear to be expressed at more similar levels between testes RNA samples of hybrids and pure species than between whole body RNA samples of hybrids and pure species. Nonetheless, at the present time, the reason for this difference in relative expression remains speculative. Figure 2. Relative expression of spermatogenesis-related transcripts in testes of D. mauritiana, D. simulans, and F1 hybrid males, bars indicating61 standard error. doi:10.1371/journal.pone.0003009.g002 PLoS ONE | www.plosone.org 3 August 2008 | Volume 3 | Issue 8 | e3009