Beyond Sperm and Male Accessory Gland Proteins: Exploring Insect Reproductive Metabolomes

Insect seminal fluid, the non-sperm component of the ejaculate, comprises a variegated set of molecules, including, but not limited to, lipids, proteins, carbohydrates, salts, hormones, nucleic acids, and vitamins. The identity and functional role of seminal fluid proteins (SFPs) have been widely investigated, in multiple species. However, most of the other small molecules in insect ejaculates remain uncharacterized. Metabolomics is currently adopted to deepen our understanding of complex biological processes and in the last 15years has been applied to answer different physiological questions. Technological advances in high-throughput methods for metabolite identification such as mass spectrometry and nuclear magnetic resonance (NMR) are now coupled to an expanded bioinformatics toolbox for large-scale data analysis. These improvements allow for the processing of smaller-sized samples and for the identification of hundreds to thousands of metabolites, not only in Drosophila melanogaster but also in disease vectors, animal, and agricultural pests. In this review, we provide an overview of the studies that adopted metabolomics-based approaches in insects, with a particular focus on the reproductive tract (RT) of both sexes and the ejaculate. Progress in the field of metabolomics will contribute not only to achieve a deeper understanding of the composition of insect ejaculates and how they are affected by endogenous and exogenous factors, but also to provide increasingly powerful tools to decipher the identity and molecular interactions between males and females during and after mating.

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Differences in Postmating Transcriptional Responses between Conspecific and Heterospecific Matings in Drosophila

Molecular Biology and Evolution ◽

10.1093/molbev/msaa264 ◽

2020 ◽

Author(s):

Yasir H Ahmed-Braimah ◽

Mariana F Wolfner ◽

Andrew G Clark

Keyword(s):

Reproductive Tract ◽

Evolutionary Dynamics ◽

Seminal Fluid ◽

Female Reproductive Tract ◽

Additional Stress ◽

Reproductive Proteins ◽

Transcriptional Responses ◽

Seminal Fluid Proteins ◽

Reproductive Genes ◽

Imd Pathway

Abstract In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins and are critical for fertilization success. Unfortunately, our understanding of the molecular interplay between female and male reproductive proteins remains inadequate. Here, we analyze the postmating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; Drosophila novamexicana females produce only ∼1% fertilized eggs in crosses with Drosophila americana males, compared to ∼98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study, we use short-read RNA sequencing to examine the evolutionary dynamics of female reproductive genes and the postmating transcriptome response in crosses within and between species. First, we found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, postmating responses in con- and heterospecific matings are largely congruent, but heterospecific matings induce expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. We also identify several genes in the JAK/STAT signaling pathway that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, we also detect it in the female head and ovaries. These results show that the female’s postmating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.

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Differences in post-mating transcriptional responses between conspecific and heterospecific matings in Drosophila

10.1101/2020.03.25.009068 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yasir H. Ahmed-Braimah ◽

Mariana F. Wolfner ◽

Andrew G. Clark

Keyword(s):

Reproductive Tract ◽

Evolutionary Dynamics ◽

Seminal Fluid ◽

Female Reproductive Tract ◽

Additional Stress ◽

Reproductive Proteins ◽

Transcriptional Responses ◽

Seminal Fluid Proteins ◽

Reproductive Genes ◽

Imd Pathway

AbstractIn many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins, and are critical for fertilization success. Unfortunately, our understanding of the molecular interplay between female and male reproductive proteins remains superficial. Here we analyze the post-mating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; D. novamexicana females produce only 1% fertilized eggs in crosses with D. americana males, compared to ~98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study we use short-read RNA sequencing to examine the evolutionary dynamics of female reproductive genes and the post-mating transcriptome response in crosses within and between species. First, we found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, post-mating responses in con- and heterospecific matings are largely congruent, but heterospecific mating induces expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. We also identify several genes in the JAK/STAT signaling pathway that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, we also detect it in the female head and ovaries. Our results show that the female’s post-mating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.

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Research gaps and new insights in the intriguing evolution of Drosophila seminal proteins

10.1101/2021.05.11.443674 ◽

2021 ◽

Author(s):

Juan Hurtado ◽

Francisca Cunha Almeida ◽

Silvina Anahí Belliard ◽

Santiago Revale ◽

Esteban Hasson

Keyword(s):

Seminal Fluid ◽

Current Knowledge ◽

Comparative Genomic ◽

Genomic Information ◽

Research Gaps ◽

Seminal Fluid Proteins ◽

Open Questions ◽

Starting Point ◽

Genomic Analyses ◽

Multiple Species

While the striking effects that seminal fluid proteins (SFPs) exert on females are fairly conserved among Diptera, they exhibit remarkable evolutionary lability. Consequently, most SFPs lack detectable homologs among the repertoire of SFPs of phylogenetically distant species. How such a rapidly changing proteome "manages" to conserve functions across taxa is a fascinating question. However, this and other pivotal aspects of SFPs' evolution remain elusive because discoveries on these proteins have been mainly restricted to the model D. melanogaster. Here, we provide an overview of the current knowledge on the inter-specific divergence of Drosophila SFPs and compile the increasing amount of relevant genomic information from multiple species. Capitalizing the accumulated knowledge in D. melanogaster, we present novel sets of high-confidence SFP candidates and transcription factors presumptively involved in regulating the expression of SFPs. We also address open questions by performing comparative genomic analyses that failed to support the existence of conserved SFPs shared by most dipterans and indicated that gene co-option is the most frequent mechanism accounting for the origin of Drosophila SFP-coding genes. We hope our update establishes a starting point to integrate, as more species are assayed for SFPs, further data and thus, to widen the understanding of the intricate evolution of these proteins.

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Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/139.1.189 ◽

1995 ◽

Vol 139 (1) ◽

pp. 189-201 ◽

Cited By ~ 15

Author(s):

A G Clark ◽

M Aguadé ◽

T Prout ◽

L G Harshman ◽

C H Langley

Keyword(s):

Reproductive Tract ◽

Seminal Fluid ◽

Glucose Dehydrogenase ◽

Natural Populations ◽

Fertilization Success ◽

Accessory Gland ◽

Male Reproductive Success ◽

Sperm Displacement ◽

Naturally Occurring ◽

Accessory Gland Protein

Abstract Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement.

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Rapid evolution of reproductive proteins in abalone and Drosophila

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2005.1793 ◽

2006 ◽

Vol 361 (1466) ◽

pp. 261-268 ◽

Cited By ~ 87

Author(s):

Tami M Panhuis ◽

Nathaniel L Clark ◽

Willie J Swanson

Keyword(s):

Protein Evolution ◽

Sexual Conflict ◽

Reproductive Tract ◽

Rapid Evolution ◽

Theoretical Data ◽

Pathogen Resistance ◽

Accessory Gland ◽

Female Reproductive Tract ◽

Reproductive Proteins ◽

Accessory Gland Proteins

Observations from different taxa, including plants, protozoa, insects and mammals, indicate that proteins involved in reproduction evolve rapidly. Several models of adaptive evolution have been proposed to explain this phenomenon, such as sexual conflict, sexual selection, self versus non-self recognition and pathogen resistance. Here we discuss the potential role of sexual conflict in the rapid evolution of reproductive genes in two different animal systems, abalone ( Haliotis ) and Drosophila . In abalone, we reveal how specific interacting sperm–egg proteins were identified and discuss this identification in the light of models for rapid protein evolution and speciation. For Drosophila , we describe the genomic approaches taken to identify male accessory gland proteins and female reproductive tract proteins. Patterns of protein evolution from both abalone and Drosophila support the predicted patterns of rapid protein evolution driven by sexual conflict. We stress however that other selective pressures may contribute to the rapid evolution that is observed. We conclude that the key to distinguishing between sexual conflict and other mechanisms of protein evolution will be an integration of genetic, experimental and theoretical data.

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A genome-wide analysis of courting and mating responses in Drosophila melanogaster females

Genome ◽

10.1139/g04-050 ◽

2004 ◽

Vol 47 (5) ◽

pp. 900-910 ◽

Cited By ~ 129

Author(s):

Mara KN Lawniczak ◽

David J Begun

Keyword(s):

Gene Expression ◽

Drosophila Melanogaster ◽

Seminal Fluid ◽

Differentially Expressed ◽

Male Reproductive Success ◽

Female Side ◽

Accessory Gland Proteins ◽

Seminal Fluid Proteins ◽

A Genome ◽

Immune Related Genes

In Drosophila melanogaster, seminal fluid proteins influence several components of female physiology and behavior, including re-mating rates, ovulation and oviposition, and sperm use. It is well-known that female flies are not simply passive vessels and that female-mediated interactions with male products are important to female (and thus male) reproductive success. While the population genetics, molecular evolution and physiological effects of seminal fluid proteins have been examined, the genetics and evolution of the female side of these post-mating interactions is unexplored in spite of work showing that female genotype and female-by-male genotype interactions are important determinants of sperm competition outcomes. Here we use microarrays to identify candidate genes involved in the female side of post-mating sexual interactions. We report the results of a whole-genome oligonucleotide chip experiment that reveals 23 genes differentially expressed between virgin females exposed and unexposed to courting males, and 38 genes differentially expressed between virgin and recently mated females. Immune related genes are overrepresented among the mating-influenced candidates. We use quantitative reverse-transcriptase PCR to independently assess gene expression changes for roughly half of the mating-affected candidate genes.Key words: reproduction, gene expression, Drosophila immune related genes, serine proteases, accessory gland proteins.

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Male Seminal Fluid Proteins Are Essential for Sperm Storage in Drosophila melanogaster

Genetics ◽

10.1093/genetics/153.2.837 ◽

1999 ◽

Vol 153 (2) ◽

pp. 837-844 ◽

Cited By ~ 9

Author(s):

Uyen Tram ◽

Mariana F Wolfner

Keyword(s):

Drosophila Melanogaster ◽

Seminal Fluid ◽

Accessory Gland ◽

Sperm Storage ◽

Competitive Pcr ◽

Seminal Fluid Proteins ◽

Pcr Assays ◽

Transgenic Drosophila ◽

Direct Counts

AbstractThe seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male’s reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.

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Endocycles support tissue growth and regeneration of the adult Drosophila accessory gland

10.1101/719013 ◽

2019 ◽

Cited By ~ 2

Author(s):

Allison M. Box ◽

Samuel Jaimian Church ◽

David Hayes ◽

Shyama Nandakumar ◽

Russell S. Taichman ◽

...

Keyword(s):

Cell Size ◽

Seminal Fluid ◽

Cell Types ◽

Accessory Gland ◽

Tissue Growth ◽

Critical Function ◽

Seminal Fluid Proteins ◽

Functional Analog ◽

Secretory Epithelial Cell ◽

Adult Drosophila

AbstractThe Drosophila melanogaster accessory gland is a functional analog of the mammalian prostate made up of two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. Here we show that similar to the mammalian prostate, this tissue grows with age. We find that the adult accessory gland grows in part via endocycles to increase DNA content and cell size, independent of mating status. The differentiated, bi-nucleated main cells remain poised to endocycle in the adult gland and upregulation of signals that promote endocycling and tissue growth are sufficient to trigger dramatic endocycling leading to increases in cell size and ploidy. The main cells of this tissue remain poised to enter the cell cycle and endocycling of main cells increases during recovery from severe tissue damage. Our data establish that the adult accessory gland is not quiescent, but instead uses endocycles to maintain the accessory gland’s critical function throughout the fruit fly’s lifespan.

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Single-nucleus transcriptomes reveal evolutionary and functional properties of cell types in the Drosophila accessory gland

Genetics ◽

10.1093/genetics/iyab213 ◽

2021 ◽

Cited By ~ 1

Author(s):

Alex C Majane ◽

Julie M Cridland ◽

David J Begun

Keyword(s):

Seminal Fluid ◽

Ejaculatory Duct ◽

Evolutionary Process ◽

Cell Types ◽

Accessory Gland ◽

Male Reproduction ◽

Seminal Fluid Proteins ◽

Cell Diversity ◽

Single Nucleus ◽

First Time

Abstract Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in post-copulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female post-copulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female post-copulatory biology, yet relatively little is known about their evolutionary properties. Here we use single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. We find that seminal fluid transcripts are strongly partitioned across the major cell types, and expression of many other genes additionally define each cell type. We also report previously undocumented diversity in main cells. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for our understanding of selection on male post-copulatory traits.

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Gene Expression and Alternative Splicing Dynamics Are Perturbed in Head Transcriptomes of Heterospecifically Mated Females

10.21203/rs.3.rs-149189/v1 ◽

2021 ◽

Author(s):

Fernando Diaz ◽

Carson Allan ◽

Therese Markow ◽

Jeremy Bono ◽

Luciano Matzkin

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Reproductive Tract ◽

Seminal Fluid ◽

Intron Retention ◽

Transcriptional Responses ◽

Seminal Fluid Proteins ◽

Genome Wide ◽

Amino Acid Balance ◽

Postmating Behavior

Abstract Background Despite the growing interest in the female side of copulatory interactions, the roles played by alternative splicing mechanisms of pre-RNA and the epistatic effects of these interactions on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). Results Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain’s physiology and female postmating behavior. Heterospecific matings often fail to activate regulation of some of these genes and induce expression of additional genes when compared with those of conspecifically-mated females. These results are consistent for all transcriptional mechanisms with some distinctions: DE genes were mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes are more related to photoreception and muscle assembly pathways. Conclusions IR seem to be an important mechanism of DE regulation during the female postmating response. While AS genes evolve at slower evolutionary rates than the genome background, DE genes evolve at much higher rates, similar or even higher than those of seminal fluid proteins, which unveil their potential role for reproductive barriers and the extent of sexual conflict.

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