RESUMEN
Sperm competition is a crucial aspect of male reproductive success in many species, including Drosophila melanogaster, and seminal fluid proteins (Sfps) can influence sperm competitiveness. However, the combined effect of environmental and genotypic variation on sperm competition gene expression remains poorly understood. Here, we used Drosophila Genetic Reference Panel (DGRP) inbred lines and manipulated developmental population density (i.e. larval density) to test the effects of genotype, environment and genotype-by-environment interactions (GEI) on the expression of the known sperm competition genes Sex Peptide, Acp36DE and CG9997. High larval density resulted in reduced adult body size, but expression of sperm competition genes remained unaffected. Furthermore, we found no significant GEI but genotypic effects in the expression of SP and Acp36DE. Our results also revealed GEI for relative competitive paternity success (second male paternity; P2), with genes' expression positively correlated with P2. Given the effect of genotype on the expression of genes, we conducted a genome-wide association study (GWAS) and identified polymorphisms in putative cis-regulatory elements as predominant factors regulating the expression of SP and Acp36DE. The association of genotypic variation with sperm competition outcomes, and the resilience of sperm competition genes' expression against environmental challenges, demonstrates the importance of genome variation background in reproductive fitness.
Asunto(s)
Drosophila melanogaster , Estudio de Asociación del Genoma Completo , Masculino , Animales , Drosophila melanogaster/genética , Semen , Genotipo , Drosophila , Larva , EspermatozoidesRESUMEN
Early lineage diversification is central to understand what mutational events drive species divergence. Particularly, gene misregulation in interspecific hybrids can inform about what genes and pathways underlie hybrid dysfunction. In Drosophila hybrids, how regulatory evolution impacts different reproductive tissues remains understudied. Here, we generate a new genome assembly and annotation in Drosophila willistoni and analyse the patterns of transcriptome divergence between two allopatrically evolved D. willistoni subspecies, their male sterile and female fertile hybrid progeny across testis, male accessory gland, and ovary. Patterns of transcriptome divergence and modes of regulatory evolution were tissue-specific. Despite no indication for cell-type differences in hybrid testis, this tissue exhibited the largest magnitude of expression differentiation between subspecies and between parentals and hybrids. No evidence for anomalous dosage compensation in hybrid male tissues was detected nor was a differential role for the neo- and the ancestral arms of the D. willistoni X chromosome. Compared to the autosomes, the X chromosome appeared enriched for transgressively expressed genes in testis despite being the least differentiated in expression between subspecies. Evidence for fine genome clustering of transgressively expressed genes suggests a role of chromatin structure on hybrid gene misregulation. Lastly, transgressively expressed genes in the testis of the sterile male progeny were enriched for GO terms not typically associated with sperm function, instead hinting at anomalous development of the reproductive tissue. Our thorough tissue-level portrait of transcriptome differentiation between recently diverged D. willistoni subspecies and their hybrids provides a more nuanced view of early regulatory changes during speciation.
Asunto(s)
Drosophila , Semen , Animales , Masculino , Femenino , Drosophila/genética , Cromosoma X , Diferenciación Celular , Transcriptoma/genética , Hibridación GenéticaRESUMEN
BACKGROUND: The genetic basis of hybrid incompatibilities is characterized by pervasive cases of gene interactions. Sex chromosomes play a major role in speciation and X-linked hybrid male sterility (HMS) genes have been identified. Interestingly, some of these genes code for proteins with DNA binding domains, suggesting a capability to act as trans-regulatory elements and disturb the expression of a large number of gene targets. To understand how interactions between trans- and cis-regulatory elements contribute to speciation, we aimed to map putative X-linked trans-regulatory elements and to identify gene targets with disrupted gene expression in sterile hybrids between the subspecies Drosophila pseudoobscura pseudoobscura and D. p. bogotana. RESULTS: We find six putative trans-regulatory proteins within previously mapped X chromosome HMS loci with sequence changes that differentiate the two subspecies. Among them, the previously characterized HMS gene Overdrive (Ovd) had the largest number of amino acid changes between subspecies, with some substitutions localized within the protein's DNA binding domain. Using an introgression approach, we detected transcriptional responses associated with a sterility/fertility Ovd allele swap. We found a network of 52 targets of Ovd and identified cis-regulatory effects among target genes with disrupted expression in sterile hybrids. However, a combined analysis of polymorphism and divergence in non-coding sequences immediately upstream of target genes found no evidence of changes in candidate regulatory proximal cis-elements. Finally, peptidases were over-represented among target genes. CONCLUSIONS: We provide evidence of divergence between subspecies within the DNA binding domain of the HMS protein Ovd and identify trans effects on the expression of 52 gene targets. Our results identify a network of trans-cis interactions with possible effects on HMS. This network provides molecular evidence of gene × gene incompatibilities as contributors to hybrid dysfunction.
Asunto(s)
Proteínas de Drosophila , Infertilidad Masculina , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Fertilidad , Hibridación Genética , Masculino , Cromosomas Sexuales , Cromosoma X/genéticaRESUMEN
BACKGROUND: Female reproductive behaviors and physiology change profoundly after mating. The control of pregnancy-associated changes in physiology and behaviors are largely hard-wired into the brain to guarantee reproductive success, yet the gene expression programs that direct neuronal differentiation and circuit wiring at the end of the sex determination pathway in response to mating are largely unknown. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. Here, we use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. RESULTS: Screening for mutations that affect the sensitivity to sex-peptide, we identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. CONCLUSIONS: These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Femenino , Masculino , Neuronas , Poro Nuclear , Péptidos , Diferenciación Sexual/genética , Conducta Sexual AnimalRESUMEN
It has long been acknowledged that changes in the regulation of gene expression may account for major organismal differences. However, we still do not fully understand how changes in gene expression evolve and how do such changes influence organisms' differences. We are even less aware of the impact such changes might have in restricting gene flow between species. Here, we focus on studies of gene expression and speciation in the Drosophila model. We review studies that have identified gene interactions in post-mating reproductive isolation and speciation, particularly those that modulate male gene expression. We also address studies that have experimentally manipulated changes in gene expression to test their effect in post-mating reproductive isolation. We highlight the need for a more in-depth analysis of the role of selection causing disrupted gene expression of such candidate genes in sterile/inviable hybrids. Moreover, we discuss the relevance to incorporate more routinely assays that simultaneously evaluate the potential effects of environmental factors and genetic background in modulating plastic responses in male genes and their potential role in speciation.
Asunto(s)
Drosophila , Expresión Génica , Especiación Genética , Animales , Drosophila/genética , Flujo Génico , Masculino , Aislamiento ReproductivoRESUMEN
Drosophila pseudoobscura pseudoobscura and Drosophila pseudoobscura bogotana are two closely related subspecies with incomplete reproductive isolation. A genome-wide comparison of expression in hybrids relative to parental subspecies has been previously used to identify genes with significant changes in expression uniquely associated with the sterile condition. The misexpression (i.e., gene expression beyond levels found in parentals) of such genes could be directly linked to the onset of sterility or could alternatively be caused by incompatibilities in a hybrid genome without a direct link to sterility. Cell adhesion was previously found to be one of the largest gene ontologies with changes in expression linked to sterility. Here we used gene expression assays in fertile backcross male progeny, along with introgression progeny in which we swap a major hybrid male sterility (HMS) allele, to generate fertile and sterile males genotypically similar to F1 sterile hybrids. We identify a cell adhesion gene (GA10921) whose change in expression is directly linked to sterility and modulated by a previously characterized HMS protein. GA10921 adds to our rather limited knowledge of changes in gene expression associated with HMS, and to the identification of gene interacting partners linked to HMS.
Asunto(s)
Adhesión Celular/genética , Drosophila/genética , Drosophila/fisiología , Infertilidad Masculina/genética , Animales , Drosophila/clasificación , Hibridación Genética , Masculino , Especificidad de la EspecieRESUMEN
Understanding the origin of species is of interest to biologist in general and evolutionary biologist in particular. Hybrid male sterility (HMS) has been a focus in studies of speciation because sterility imposes a barrier to free gene flow between organisms, thus effectively isolating them as distinct species. In this review, I focus on the role of differential gene expression in HMS and speciation. Microarray and qPCR assays have established associations between misregulation of gene expression and sterility in hybrids between closely related species. These studies originally proposed disrupted expression of spermatogenesis genes as a causative of sterility. Alternatively, rapid genetic divergence of regulatory elements, particularly as they relate to the male sex (fast-male evolution), can drive the misregulation of sperm developmental genes in the absence of sterility. The use of fertile hybrids (both backcross and F1 progeny) as controls has lent support to this alternative explanation. Differences in gene expression between fertile and sterile hybrids can also be influenced by a pattern of faster evolution of the sex chromosome (fast-X evolution) than autosomes. In particular, it would be desirable to establish whether known X-chromosome sterility factors can act as trans-regulatory drivers of genome-wide patterns of misregulation. Genome-wide expression studies coupled with assays of proxies of sterility in F1 and BC progeny have identified candidate HMS genes but functional assays, and a better phenotypic characterization of sterility phenotypes, are needed to rigorously test how these genes might contribute to HMS.
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Quimera/genética , Especiación Genética , Infertilidad Masculina/genética , Animales , Evolución Biológica , Drosophila/genética , Fertilidad/genética , Expresión Génica/genética , Expresión Génica/fisiología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genoma , Masculino , Cromosomas Sexuales , Espermatogénesis/genéticaRESUMEN
A major evolutionary change in the lineage ancestral to humans, chimpanzee and gorilla (HCG) has been the embedding of the embryo into maternal tissue. Thus, the first layer of cells (trophoblast) to differentiate after fertilization must adapt to invade the uterus. Such event would likely leave signatures of positive selection at genes with roles in embryo implantation. Here, 163 pregnancy implantation genes are tested for evidence of adaptive diversification in the ancestral lineage to HCG. Two immune system genes, HLA-E and KIR2DL4 showed evidence of positive selection. Some of the positive selected sites involve amino acid substitution with predicted damaging effects on protein function, thus highlighting the possibility of antagonistic pleiotropic effects. Selection at a gene coding for a receptor expressed in uterine cells (KIR) that interacts with trophoblast human leukocyte antigen (HLA) genes suggests a main role for immunological adaptations in embryo deep invasion of the maternal endometrium.
Asunto(s)
Implantación del Embrión , Endometrio/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Embarazo/genética , Primates/genética , Receptores KIR2DL4/genética , Sustitución de Aminoácidos , Animales , Evolución Molecular , Femenino , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Primates/embriología , Receptores KIR2DL4/metabolismo , Selección Genética , Trofoblastos/fisiología , Antígenos HLA-ERESUMEN
BACKGROUND: The biological concept of species stresses the importance of understanding what mechanisms maintain species reproductively isolated from each other. Often such mechanisms are divided into premating and postmating, with the latest being the result of either prezygotic or postzygotic isolation barriers. Drosophila willistoni quechua and Drosophila willistoni willistoni are two subspecies that experience reproductive isolation. When a D. w. quechua female is crossed with a D. w. willistoni male, the hybrid males (F1QW) are unable to father progeny; however, the reciprocal cross produces fertile hybrids. Thus, the mechanism of isolation is unidirectional hybrid male sterility. However, the sterile F1QW males contain large amounts of motile sperm. Here we explore whether pre-copulatory or post-copulatory pre-zygotic mechanisms serve as major deterrents in the ability of F1QW males to father progeny. RESULTS: Comparisons of parental and hybrid males copulation durations showed no significant reduction in copulation duration of F1QW males. Interrupted copulations of the parental species confirmed that sperm transfer occurs before the minimum copulation duration registered for F1QW males. However, we found that when females mate with F1QW males, sperm is not present inside the female storage organs and that the lack of sperm in storage is due to failure to transfer sperm rather than spillage or active sperm dumping by females. CONCLUSIONS: Sterility of F1QW hybrid males is primarily driven by their inability to transfer sperm during copulation.
Asunto(s)
Drosophila/clasificación , Drosophila/fisiología , Hibridación Genética , Animales , Copulación , Drosophila/genética , Femenino , Fertilidad , Especiación Genética , Masculino , Reproducción , Aislamiento Reproductivo , EspermatozoidesRESUMEN
The role of sexual selection in driving the rapid evolution of male reproductive proteins has been tested in a wide variety of organisms. Sperm competition is a form of postmating sexual selection that can contribute to reproductive isolation between species by biasing the proportion of progeny fathered by conspecific over heterospecific males. This phenomenon is known as conspecific sperm precedence (CSP). A previous quantitative trait loci study between Drosophila simulans and D. sechellia identified a locus associated with CSP within the second chromosome centered at the 53 cytogenetic map position. Male accessory gland proteins (ACPs) are associated with triggering postmating physiological responses in D. melanogaster females that can contribute to differential male reproductive success. Moreover, a large number of ACPs evolve rapidly and under positive selection among closely-related species of Drosophila. Here we have sequenced five candidate Acp genes (Acp53C14a, Acp53C14b, Acp53C14c, Acp53Ea and Acp54A1) within the previously mapped D. simulans-D. sechellia CSP locus from different D. simulans and D. sechellia strains. Polymorphism data analysis shows evidence of a selective sweep at Acp53Ea within D. simulans. In the context of CSP, the combined use of polymorphism and interspecies sequence divergence shows that Acp53C14c gene tree topology separates D. simulans and D. sechellia. Moreover, Acp53C14c is the only gene showing evidence of positive selection with five fixed amino acid substitutions between species. Our results highlight Acp53C14c as a candidate gene for future gene targeting studies to elucidate its role in CSP between D. simulans and D. sechellia.
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Drosophila/genética , Sitios de Carácter Cuantitativo , Selección Genética , Espermatozoides/fisiología , Sustitución de Aminoácidos , Animales , Masculino , Filogenia , Polimorfismo Genético , Reproducción/genética , Aislamiento Reproductivo , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Seminal fluid protein (Sfp) genes show, in general, a higher rate of sequence divergence than genes from other categories, which is often attributed to forms of postcopulatory sexual selection or sexual conflict. Recently, the relaxation of selective constraints has been proposed as an alternative explanation for the rapid sequence evolution of Sfps and other genes with sex-limited expression. The expression of Sfp genes is a likely target of selection, but the evolution of differences in their expression levels is less understood. Here, we explore both polymorphism and divergence in Sfp gene expression between Drosophila melanogaster and Drosophila simulans, how selection might have influenced their expression, and whether changes in expression might trigger the evolution of reproductive isolating barriers. In our analysis, Sfp genes showed higher divergence, but not higher polymorphism, in expression than the average male reproductive glands gene. Sfp genes with reproductive-tissue-specific expression were enriched for both directional and stabilizing selection, while relaxed selection was the predominant mode of evolution among Sfp genes with any other nonreproductive tissue-specific or nontissue-specific expression. The knockdown of single genes known to affect intraspecific sperm competition, and with patterns of expression divergence and polymorphism suggestive of directional selection, was not enough to break down postmating reproductive isolation barriers between species. Our results identify the expression of male-specific Sfp genes as an enriched target of selection and suggest a complex molecular relationship between postcopulatory sexual selection on a single gene's expression and its effect on the onset of speciation.
Asunto(s)
Drosophila melanogaster , Evolución Molecular , Aislamiento Reproductivo , Animales , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Plasma Seminal/genética , Selección Genética , Polimorfismo Genético , Drosophila simulans/genética , Femenino , Semen , Drosophila/genética , Drosophila/fisiologíaRESUMEN
BACKGROUND: Sperm and testes-expressed Adam genes have been shown to undergo bouts of positive selection in mammals. Despite the pervasiveness of positive selection signals, it is unclear what has driven such selective bouts. The fact that only sperm surface Adam genes show signals of positive selection within their adhesion domain has led to speculation that selection might be driven by species-specific adaptations to fertilization or sperm competition. Alternatively, duplications and neofunctionalization of Adam sperm surface genes, particularly as it is now understood in rodents, might have contributed to an acceleration of evolutionary rates and possibly adaptive diversification. RESULTS: Here we sequenced and conducted tests of selection within the adhesion domain of sixteen known sperm-surface Adam genes among five species of the Mus genus. We find evidence of positive selection associated with all six Adam genes known to interact to form functional complexes on Mus sperm. A subset of these complex-forming sperm genes also displayed accelerated branch evolution with Adam5 evolving under positive selection. In contrast to our previous findings in primates, selective bouts within Mus sperm Adams showed no associations to proxies of sperm competition. Expanded phylogenetic analysis including sequence data from other placental mammals allowed us to uncover ancient and recent episodes of adaptive evolution. CONCLUSIONS: The prevailing signals of rapid divergence and positive selection detected within the adhesion domain of interacting sperm Adams is driven by duplications and potential neofunctionalizations that are in some cases ancient (Adams 2, 3 and 5) or more recent (Adams 1b, 4b and 6).
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Proteínas ADAM/genética , Evolución Molecular , Ratones/genética , Espermatozoides/metabolismo , Proteínas ADAM/química , Proteínas ADAM/clasificación , Animales , Fertilización , Duplicación de Gen , Masculino , Ratones/clasificación , Filogenia , Estructura Terciaria de Proteína , Selección Genética , Especificidad de la EspecieRESUMEN
Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.
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Drosophila/clasificación , Drosophila/genética , Evolución Molecular , Genes de Insecto/genética , Genoma de los Insectos/genética , Genómica , Filogenia , Animales , Codón/genética , Elementos Transponibles de ADN/genética , Drosophila/inmunología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Orden Génico/genética , Genoma Mitocondrial/genética , Inmunidad/genética , Familia de Multigenes/genética , ARN no Traducido/genética , Reproducción/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Sintenía/genéticaRESUMEN
Nuclear pore proteins (Nups) prominently are among the few genes linked to speciation from hybrid incompatibility in Drosophila. These studies have focused on coding sequence evolution of Nup96 and Nup160 and shown evidence of positive selection driving nucleoporin evolution. Intriguingly, channel Nup54 functionality is required for neuronal wiring underlying the female post-mating response induced by male-derived sex-peptide. A region of rapid evolution in the core promoter of Nup54 suggests a critical role for general transcriptional regulatory elements at the onset of speciation, but whether this is a general feature of Nup genes has not been determined. Consistent with findings for Nup54, additional channel Nup58 and Nup62 promoters also rapidly accumulate insertions/deletions (indels). Comprehensive examination of Nup upstream regions reveals that core Nup complex gene promoters accumulate indels rapidly. Since changes in promoters can drive changes in expression, these results indicate an evolutionary mechanism driven by indel accumulation in core Nup promoters. Compensation of such gene expression changes could lead to altered neuronal wiring, rapid fixation of traits caused by promoter changes and subsequently the rise of new species. Hence, the nuclear pore complex may act as a nexus for species-specific changes via nucleo-cytoplasmic transport regulated gene expression.
Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Animales , Masculino , Femenino , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular/genética , Drosophila/genética , Drosophila/metabolismo , Mutación INDELRESUMEN
BACKGROUND: The rapid evolution of seminal fluid proteins (SFPs) has been suggested to be driven by adaptations to postcopulatory sexual selection (e.g. sperm competition). However, we have recently shown that most SFPs evolve rapidly under relaxed selective pressures. Given the role of SFPs in competition for fertilization phenotypes, like the ability to transfer and store sperm and the modulation of female receptivity and ovulation, the prevalence of selectively relaxed SFPs appears as a conundrum. One possible explanation is that selection on SFPs might be relaxed in terms of protein amino acid content, but adjustments of expression are essential for post-mating function. Interestingly, there is a general lack of systematic implementation of gene expression perturbation assays to monitor their effect on phenotypes related to sperm competition. RESULTS: We successfully manipulated the expression of 16 SFP encoding genes using tissue-specific knockdowns (KDs) and determined the effect of these genes' perturbation on three important post-mating phenotypes: female refractoriness to remating, defensive (P1), and offensive (P2) sperm competitive abilities in Drosophila melanogaster. Our analyses show that KDs of tested SFP genes do not affect female refractoriness to remating and P2, however, most gene KDs significantly decreased P1. Moreover, KDs of SFP genes that are selectively constrained in terms of protein-coding sequence evolution have lower P1 than KDs of genes evolving under relaxed selection. CONCLUSIONS: Our results suggest a more predominant role, than previously acknowledged, of variation in gene expression than coding sequence changes on sperm competitive ability in D. melanogaster.
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Proteínas de Drosophila , Drosophila melanogaster , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Expresión Génica , Aptitud Genética , Masculino , Espermatozoides/metabolismoRESUMEN
The study of human neurological infection faces many technical and ethical challenges. While not as common as mammalian models, the use of Drosophila (fruit fly) in the investigation of virus-host dynamics is a powerful research tool. In this review, we focus on the benefits and caveats of using Drosophila as a model for neurological infections and neuroimmunity. Through the examination of in vitro, in vivo and transgenic systems, we highlight select examples to illustrate the use of flies for the study of exogenous and endogenous viruses associated with neurological disease. In each case, phenotypes in Drosophila are compared to those in human conditions. In addition, we discuss antiviral drug screening in flies and how investigating virus-host interactions may lead to novel antiviral drug targets. Together, we highlight standardized and reproducible readouts of fly behaviour, motor function and neurodegeneration that permit an accurate assessment of neurological outcomes for the study of viral infection in fly models. Adoption of Drosophila as a valuable model system for neurological infections has and will continue to guide the discovery of many novel virus-host interactions.
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Enfermedades Transmisibles , Proteínas de Drosophila , Virus , Animales , Enfermedades Transmisibles/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Humanos , Mamíferos/metabolismo , Interferencia de ARN , Virus/metabolismoRESUMEN
Male sex genes have shown a pattern of rapid interspecies divergence at both the coding and gene expression level. A common outcome from crosses between closely-related species is hybrid male sterility. Phenotypic and genetic studies in Drosophila sterile hybrid males have shown that spermatogenesis arrest is postmeiotic with few exceptions, and that most misregulated genes are involved in late stages of spermatogenesis. Comparative studies of gene regulation in sterile hybrids and parental species have mainly used microarrays providing a whole genome representation of regulatory problems in sterile hybrids. Real-time PCR studies can reject or reveal differences not observed in microarray assays. Moreover, differences in gene expression between samples can be dependant on the source of RNA (e.g., whole body vs. tissue). Here we survey expression in D. simulans, D. mauritiana and both intra and interspecies hybrids using a real-time PCR approach for eight genes expressed at the four main stages of sperm development. We find that all genes show a trend toward under expression in the testes of sterile hybrids relative to parental species with only the two proliferation genes (bam and bgcn) and the two meiotic class genes (can and sa) showing significant down regulation. The observed pattern of down regulation for the genes tested can not fully explain hybrid male sterility. We discuss the down regulation of spermatogenesis genes in hybrids between closely-related species within the contest of rapid divergence experienced by the male genome, hybrid sterility and possible allometric changes due to subtle testes-specific developmental abnormalities.
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Drosophila/genética , Espermatogénesis/genética , Animales , Regulación hacia Abajo , Drosophila/crecimiento & desarrollo , Drosophila/fisiología , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Variación Genética , Hibridación Genética , Infertilidad Masculina/genética , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
The recent completion of genome sequencing of 12 species of Drosophila has provided a powerful resource for hypothesis testing, as well as the development of technical tools. Here we take advantage of genome sequence data from two closely related species of Drosophila, Drosophila simulans and Drosophila sechellia, to quickly identify candidate molecular markers for genotyping based on expected insertion or deletion (indel) differences between species. Out of 64 candidate molecular markers selected along the second and third chromosome of Drosophila, 51 molecular markers were validated using PCR and gel electrophoresis. We found that the 20% error rate was due to sequencing errors in the genome data, although we cannot rule out possible indel polymorphisms. The approach has the advantage of being affordable and quick, as it only requires the use of bioinformatics tools for predictions and a PCR and agarose gel based assay for validation. Moreover, the approach could be easily extended to a wide variety of taxa with the only limitation being the availability of complete or partial genome sequence data.
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Drosophila/genética , Marcadores Genéticos/genética , Genoma de los Insectos/genética , Tipificación Molecular/métodos , Animales , Secuencia de Bases , ADN/química , ADN/genética , Drosophila/clasificación , Proteínas de Drosophila/genética , Electroforesis en Gel de Agar , Variación Genética , Genotipo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.
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Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/fisiología , Fertilidad/fisiología , Fertilización/fisiología , Cigoto/fisiología , Animales , Femenino , Expresión Génica , Masculino , EspermatozoidesRESUMEN
Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra- and interspecific sequence information, along with genomics and functional data, we examine the molecular evolution of approximately 300 SFPs in Drosophila. We found that 50-57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7-12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35-37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. Our results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution.