Gene duplication is considered a fundamental process in the generation of genes with novel functions and ultimately the evolution of biological diversity . A wide variety of evolutionary and comparative genomic analyses have been used to infer the origins and fates of newly created genes [2–5]. While more limited in scope, molecular genetic and functional studies [6–9] have provided new insights into the role gene duplication has played in generating novel functions. In Drosophila, comparative genomic analyses have revealed an enrichment of rapidly evolving or lineage specific gene families associated with reproduction . While the functional significance of this remains to be fully determined, the trend can be explained, in part, by creation of male-biased genes through retrotransposition. Recent studies have demonstrated that retrogenes in both Drosophila [11, 12] and primates [13–16] tend to acquire testis-specific expression. However, we still have limited understanding of why new genes frequently acquire testis expression and whether testis-specific expression is obtained prior to, or following, acquisition of functionality. Nonetheless, several studies suggest that newly generated genes and gene families have had a functional impact on spermatogenesis and on the evolution of sperm [8, 12, 17].
Although new testis-specific genes might be expected to have gained functions in spermatogenesis, there are a limited number of characterized examples of such genes in Drosophila. One example is the strict paternal-effect gene ms(3)K81 (termed "K81"), a gene created by retrotransposition prior to the divergence of the melanogaster subgroup . In wild-type eggs fertilized by sperm from K81 males, paternal chromosomes systematically fail to properly separate sister chromatids during the first zygotic division leading to lethality early in embryogenesis . Interestingly, K81 is expressed only in primary spermatocytes where it presumably regulates aspects of spermatogenesis required for proper sperm function in the egg. A second example is mojoless (mjl), created approximately 50mya through the retrotransposition of shaggy (sgg), a glycogen synthase kinase-3 encoding gene . RNAi knockdown of mjl resulted in loss of the male germ line and male sterility. Although not yet ascribed a characterized function in the testis or sperm, a third gene, Sdic, is a newly created gene encoding a protein present in the sperm tail . Sdic is an unusual case of an X-linked chimeric gene specific to Drosophila melanogaster that was created through the duplication of annexinX (AnnX) and subsequent fusion with Cdic, a cytoplasmic dynein gene.
The application of mass spectrometry (MS) to the study of sperm has provided our first insights into the dynamic role new gene creation has played in shaping the constituents of the sperm proteome  and has raised intriguing questions about how these new genes have impacted the molecular evolution of sperm. For example, analysis of the Drosophila sperm proteome revealed 3 novel sperm genes specific to D. melanogaster and a further 4 sperm genes created through retrotransposition during Drosophila evolution . Amongst these is the retrogene CG13340, which encodes S-LAP 7, examined in this study. It is noteworthy that these new sperm components were found to be proteins across diverse functional classes, and both rapidly evolving (in the case of protamine genes, Mst35a and Mst35b) and highly conserved (X-linked Tektin gene cluster).
Many proteolysis-related genes (including peptidases, proteases and inhibitors of proteolysis) have reproductive functions across diverse taxa, including insects [19–21] and mammals . Amongst these, proteolysis-related genes expressed in the Drosophila accessory gland have been particularly well studied in terms of their mediation of a wide range of effects on females (see  for a recent review). Proteins with proteolytic activity are also found in the Drosophila female reproductive tract and a subset of these are encoded by recently created genes . In both mammals and Drosophila, the genes involved in reproductive proteolytic pathways or their targets have been demonstrated to evolve rapidly [22, 25, 26] and it is hypothesized that this is due to coevolutionary forces associated with sexual conflict (either between males and females or males and males) [27, 28]. These observations have been extended to the sperm proteome where an intensified signature of positive selection was observed for membrane and acrosomal sperm proteins that included a diverse set of metalloproteases and protease inhibitors .
Although well documented in reproductive tissues generally, only recently have proteolytic and related genes been shown to be present in spermatozoa [18, 29–31]. The functional significance and role in fertilization of these classes of proteins is unknown, but demonstration of their presence and activity in sperm is a necessary step towards more targeted studies. Here we present a detailed evolutionary and functional characterization of a family of eight Drosophila sperm leucyl aminopeptidases that we have termed Sperm-LAPs (S-LAP 1-8). Computational annotation places the S-LAPs in the M17 family of leucyl aminopeptidases . Of the 10 annotated leucyl aminopeptidases in the Drosophila genome, the S-LAPs are specifically expressed in the testis and all encode proteins incorporated in mature sperm [18, 33]. Here we describe results of detailed comparative genomic analyses that demonstrate an expansion and diversification of the S-LAP gene family during Drosophila evolution. We also provide (i) an independent proteomic analysis confirming the presence of S-LAPs in sperm, (ii) a biochemical analysis of S-LAPs quantity and abundance in sperm, (iii) data indicating that S-LAPs are expressed specifically in the testis and (iv) evidence confirming S-LAP enzymatic activity supporting their functional annotation. This striking specificity of expression and cellular location suggests an unknown functional requirement for aminopeptidases in sperm. Furthermore, evolutionary diversification at the active site indicates that some S-LAPs have lost enzymatic activity and that the putative neofunctionalization of these non-enzymatic S-LAPs may be related to the subsequent expansion of the gene family. These findings thus support a scenario where functional diversification within a gene family may promote further evolutionary changes in gene family composition through the selective retention of newly created genes.