Gene Expression Disruptions of Organism versus Organ in Drosophila Species Hybrids Daniel J. Catron, Mohamed A. F. Noor* Biology Department, Duke University, Durham, North Carolina, United States of America Abstract Hybrid dysfunctions, such as sterility, may result in part from disruptions in the regulation of gene expression. Studies of hybrids within the Drosophila simulans clade have reported genes expressed above or below the expression observed in their parent species, and such misexpression is associated with male sterility in multigenerational backcross hybrids. However, these studies often examined whole bodies rather than testes or had limited replication using less-sensitive but global techniques. Here, we use a new RNA isolation technique to re-examine hybrid gene expression disruptions in both testes and whole bodies from single Drosophila males by real-time quantitative RT-PCR. We find two early-spermatogenesis transcripts are underexpressed in hybrid whole-bodies but not in assays of testes alone, while two late-spermatogenesis transcripts seem to be underexpressed in both whole-bodies and testes alone. Although the number of transcripts surveyed is limited, these results provide some support for a previous hypothesis that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes. Citation: Catron DJ, Noor MAF (2008) Gene Expression Disruptions of Organism versus Organ in Drosophila Species Hybrids. PLoS ONE 3(8): e3009. doi:10.1371/ journal.pone.0003009 Editor: Adam K. Chippindale, Queens University, Canada Received June 11, 2008; Accepted July 31, 2008; Published August 20, 2008 Copyright: 2008 Catron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided by National Science Foundation grants 0509780 and 0715484 and National Institutes of Health grant GM076051. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: NOOR@DUKE.EDU Introduction Hybrid dysfunctions, such as sterility or inviability, result from failed (or novel deleterious) interactions between the genomes of the two parent species. Although conclusive evidence is still lacking, several recent studies have suggested that disruptions in gene expression may be one source for these failed interactions [for reviews, see 1,2] [but see 3]. For example, disruptions in expression of Xmrk-2 in some backcross hybrids of Xiphophorus maculates and X. helleri cause spots on their dorsal fins to spontaneously develop malignant melanomas [4,5]. Theoretical investigations have also suggested that interspecies hybrids can have low fitness where natural selection has altered interacting molecules, such as the binding affinity between transcription factors and DNA binding sites, independently between the two parent species [6–8]. Disruptions in gene expression have been examined extensively in hybrids of the genus Drosophila [e.g., 9,10], and particularly within the D. simulans clade (D. simulans, D. mauritiana, D. sechellia). Recent studies have used microarrays to examine disruptions in gene expression in male hybrids of D. simulans clade species [11– 13]. All three studies found many genes severely underexpressed in the hybrids relative to the pure species, and these genes were disproportionately associated with spermatogenesis or other male- specific phenotypes. Michalak and Noor [14] further found that sterility and underexpression of five transcripts were strongly correlated in fifth generation backcross hybrids of D. simulans and D. mauritiana, and a recent study found that one of these transcripts appears to be directly involved in incompatibilities leading to hybrid sterility [15]. Thus, it is possible that misexpression of male- fertility-essential genes involved in spermatogenesis caused sterility in these hybrids. Underexpressed genes also appear to be more rapidly evolving than genes expressed normally in hybrids [16]. Finally, Moehring et al. [13] overlaid their misexpression results onto part of a known spermatogenesis pathway for D. melanogaster [see Figure 1, adapted from 17,18] and found many late-stage downstream loci exhibiting misexpression (e.g., don juan, gonadal, Mst84D, Mst98Ca, Mst98Cb, Mst87D), whereas relatively few early- stage loci were misexpressed. This finding may suggest that the spermatogenesis regulatory pathway could be disrupted at a particular stage. However, some of the studies described above [12,13] used whole adult bodies for investigating hybrid misexpression, and it is unclear if this approach was misleading. If there were tissue- specific hybrid expression disruptions, then testes-specific mis- expression may have been either missed or erroneously inferred (by disruptions in other tissues of male-related transcripts). One approach to address this concern would be to focus expression assays on testes alone, as done by Haerty and Singh [11]. However, Haerty and Singh [11] examined hybrid expression via microarrays designed with probes from a related species (D. melanogaster), which has been shown to be less-sensitive [13,19], and they used both extensive pooling across individuals and an RNA amplification step in their assays. While theirs was a good first approach, a logical next step would be to use biologically replicated quantitative real-time PCRs on cDNA pools derived from testes of single flies to examine the extent of misexpression. Our experience has shown that real-time PCR will sometimes identify expression differences not observed via microarray analyses (e.g., hybrid misexpression of always early: [20]). PLoS ONE | www.plosone.org 1 August 2008 | Volume 3 | Issue 8 | e3009