Neofunctionalization of young duplicate genes in Drosophila

Gene duplication is a key source of genetic innovation that plays a role in the evolution of phenotypic complexity. Although several evolutionary processes can result in the long-term retention of duplicate genes, their relative contributions in nature are unknown. Here we develop a phylogenetic approach for comparing genome-wide expression profiles of closely related species to quantify the roles of conservation, neofunctionalization, subfunctionalization, and specialization in the preservation of duplicate genes. Application of our method to pairs of young duplicates in Drosophila shows that neofunctionalization, the gain of a novel function in one copy, accounts for the retention of almost two-thirds of duplicate genes. Surprisingly, novel functions nearly always originate in younger (child) copies, whereas older (parent) copies possess functions similar to those of ancestral genes. Further examination of such pairs reveals a strong bias toward RNA-mediated duplication events, implicating asymmetric duplication and positive selection in the evolution of new functions. Moreover, we show that young duplicate genes are expressed primarily in testes and that their expression breadth increases over evolutionary time. This finding supports the “out-of-testes” hypothesis, which posits that testes are a catalyst for the emergence of new genes that ultimately evolve functions in other tissues. Thus, our study highlights the importance of neofunctionalization and positive selection in the retention of young duplicates in Drosophila and illustrates how duplicates become incorporated into novel functional networks over evolutionary time.Gene duplication produces two copies of an existing gene. Evolutionary theory predicts that functional redundancy of duplicate genes causes one copy to undergo a brief period of relaxed selection after duplication (1). In nearly all cases, this should result in an accumulation of deleterious mutations and pseudogenization of the copy within a few million years (2). However, most sequenced eukaryotic genomes contain many functional duplicates, some of which are hundreds of millions of years old (3–8), suggesting that duplicate genes play important roles in evolution.Four processes can result in the evolutionary preservation of duplicate genes: conservation, neofunctionalization, subfunctionalization, and specialization. Under conservation, ancestral functions are maintained in both copies, likely because increased gene dosage is beneficial (1). Under neofunctionalization, one copy retains its ancestral functions, and the other acquires a novel function (1). Under subfunctionalization, mutations damage different functions of each copy, such that both copies are required to preserve all ancestral gene functions (9, 10). Finally, under specialization, subfunctionalization and neofunctionalization act in concert, producing two copies that are functionally distinct from each other and from the ancestral gene (11). Theoretical work has shown that different conditions can result in the retention of duplicate genes by any one of these processes (9, 12–17), and empirical studies have uncovered numerous examples of each (11, 18–23).However, no genome-wide studies have attempted to distinguish among these processes and, thus, their relative roles in nature remain unknown. One difficulty of such a study is defining biological function on a genomic scale. To address this problem, we used relative gene expression levels in different tissues (i.e., gene expression profiles) as proxies for function. Gene expression profiles are ideal for assessing biological function because of the availability of high-throughput expression data for multiple tissues in a number of species, correlations to different measures of gene function (24–27), and simple quantitative interpretation relative to alternative functional metrics such as protein structure or interaction networks. A second obstacle to studying evolutionary processes underlying the retention of duplicate genes is the lack of methods for distinguishing among processes. To disentangle these evolutionary processes, we developed a phylogenetic approach for comparing expression divergence between duplicate genes (parent and child copies) in one species and their ancestral single-copy ortholog in a closely related sister species. Our approach combines gene expression profiles with phylogenetic relationships among gene copies to classify the evolutionary processes driving the preservation of young duplicate genes.     

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Ohnologs are overrepresented in pathogenic copy number mutations

A number of rare copy number variants (CNVs), including both deletions and duplications, have been associated with developmental disorders, including schizophrenia, autism, intellectual disability, and epilepsy. Pathogenicity may derive from dosage sensitivity of one or more genes contained within the CNV locus. To understand pathophysiology, the specific disease-causing gene(s) within each CNV need to be identified. In the present study, we test the hypothesis that ohnologs (genes retained after ancestral whole-genome duplication events, which are frequently dosage sensitive) are overrepresented in pathogenic CNVs. We selected three sets of genes implicated in copy number pathogenicity: (i) genes mapping within rare disease-associated CNVs, (ii) genes within de novo CNVs under negative genetic selection, and (iii) genes identified by clinical array comparative genome hybridization studies as potentially pathogenic. We compared the proportion of ohnologs between these gene sets and control genes, mapping to CNVs not known to be disease associated. We found that ohnologs are significantly overrepresented in genes mapping to pathogenic CNVs, irrespective of how CNVs were identified, with over 90% containing an ohnolog, compared with control CNVs >100 kb, where only about 30% contained an ohnolog. In some CNVs, such as del15p11.2 (CYFIP1) and dup/del16p13.11 (NDE1), the most plausible prior candidate gene was also an ohnolog, as were the genes VIPR2 and NRXN1, each found in short CNVs containing no other genes. Our results support the hypothesis that ohnologs represent critical dosage-sensitive elements of the genome, possibly responsible for some of the deleterious phenotypes observed for pathogenic CNVs and as such are readily identifiable candidate genes for further study.Copy number variants (CNVs) are microdeletions or microduplications of segments of the genome, ranging from a few hundred base pairs to several megabases (1). CNVs include common polymorphic variants segregating in the population and rare mutations which can either be inherited or occur de novo. In human disease, the discovery and ability to characterize CNVs has expanded the range of known pathogenic genome variants, especially for common, complex neurodevelopmental disorders—autism spectrum disorders (ASD), intellectual disability, schizophrenia, epilepsy, attention deficit hyperactivity disorder (ADHD), and speech and language delay (2–6).Deleterious mutations causing neurodevelopmental disorders are under strong negative genetic selection pressure (7). The majority of pathogenic CNV mutations are heterozygous deletions or single-copy duplications, i.e., increase or decrease gene dosage, and can be considered distinct from pathogenic loss of function mutations, which are not dosage effects but complete loss of function. For example, there are recessive homozygous deletions, such as nephronophthisis, in which gene deletion may be benign in heterozygous form but deleterious when homozygous (8). There are also allelic series, such as NRXN1 (neurexin 1) gene deletions, where a heterozygous deletion is a risk factor for neurodevelopmental disorders (9) and homozygous deletion causes Pitt–Hopkins-like syndrome (Online Mendelian Inheritance in Man database, www.omim.org), a severe, syndromic form of neurodisability (10). They also tend to be pleiotropic, i.e., show association across neurodevelopmental phenotypes, and also have non-CNS phenotypes, including obesity and cardiac anomalies (11).It is possible that genomic deletions are more likely to cause dosage sensitivity compared with duplications because the fold change is greater for deletions, i.e., there is a bigger proportional effect on dosage. There is evidence for this from nonallelic homologous recombination-mediated CNVs, such as 15q11.2 and 15q13.3, where the reciprocal mutational event should generate an equal number of deletions and duplications. The 15q11.2 duplication appears to have a benign or at least milder phenotype compared with the reciprocal deletion, and the population frequency of the duplication (0.39%) is greater than the deletion (0.18%), indicating the latter is under stronger negative genetic selection (5). On the other hand, overexpression in some developmental systems could have a bigger phenotypic effect than reduction in expression, and in some cases, both deletion and duplication are pathogenic, such as distal 16p11.2 (12).Pathogenic CNVs provide an opportunity for stratified medicine, because as high or moderate risk factors they may allow the selection of patients for specific interventions, such as targeted pharmacotherapy, and they may clarify disease pathophysiology and the relationship between different diagnostic categories. However, it is not straightforward to identify the specific genes that give rise to the observed phenotypes. Thus, although a few pathogenic CNVs can be mapped to a single gene, such as NRXN1 or VIPR2 (vasoactive intestinal peptide receptor 2), providing a direct route to the pathophysiology of disease, most pathogenic CNVs are large, covering several megabases, and typically delete or duplicate multiple genes (13, 14).Phenotypes are also complex. For example, 22q11.2 deletion syndrome consists of variable combinations of over 190 phenotypic characteristics, including conotruncal heart and cleft palate defects in about 70% of cases (15). Identifying the deleted genes involved in neurodevelopmental phenotypes such as psychosis has proven difficult as there are a number of plausible candidates involved in neurotransmission, neuronal development, myelination, microRNA processing, and posttranslational protein modifications (16), including catechol-(O)-methyl transferase (17) and proline dehydrogenase (18).Various strategies are possible to try and understand the relationship between specific genes and phenotypes within pathogenic CNVs. These include deletion mapping, i.e., searching for critical disease-associated intervals within larger CNV boundaries to narrow the number of genes involved; searching for inactivating mutations in single genes which carry the same phenotype as the pathogenic CNV (19); the use of bioinformatic information such as gene function and known disease associations; and the analysis of deleterious mouse knockout phenotypes to identify the most likely candidate gene(s) for a given CNV phenotype (20). Here we explore a unique evolutionary approach that identifies genes within pathogenic CNVs that have patterns of gene duplication characteristic of dosage-balanced genes (21).Dosage balance may exist between two or more genes due to stoichiometric constraints of the protein complexes or the biochemical pathways in which the gene products participate (22, 23). Changes in gene copy number through gene duplication or loss lead to increases or decreases of gene product in the cell (24, 25). In the case of dosage-balanced genes such changes are deleterious and are removed by natural selection. Thus, a general feature of dosage-balanced genes is their low duplicability (26). An exception to this is duplication through whole-genome duplication (WGD) which increases all genes equally, maintaining the relative frequencies. Thus, WGD creates a unique opportunity for the duplication of dosage-balanced genes because it guarantees the simultaneous duplication of all components of a balanced set (26–28). Furthermore, once the genes have been duplicated, subsequent loss of individual genes would result in a dosage imbalance, thus leading to biased retention of dosage-balanced genes.Two WGD events occurred early in the vertebrate lineage, generating initially tetraploid genomes. The genome duplication events were followed by extensive chromosomal rearrangement and loss of many duplicate copies (29–32). The 20–30% of the protein-coding genes in the human genome that can be traced back to these events (21, 32) have been postulated to have accelerated the evolution of vertebrate complexity, as they are enriched for developmental genes (33–35) and members of protein complexes (36).Duplicated genes derived from WGD are known as “ohnologs” (37). Consistent with expectations, ohnologs have many characteristics of dosage-balanced genes. Mammalian ohnologs are more essential (i.e., knockout of one copy is more likely to result in sterility or inviability) than paralogs generated by small-scale duplication (SSD) and have comparable essentiality to singleton genes (36), i.e., copy number change is under negative selection. The overrepresentation of ohnologs in protein complexes is also consistent with the gene dosage balance hypothesis, which states that the stoichiometry of members of multisubunit complexes affects the function of the whole because of the kinetics and mode of assembly. Changing the dosage of a structural gene (for example by deletion or duplication) changes its expression in proportion to copy number (28, 38). If the product of a gene that changes in copy number belongs to a large protein complex, this may generate stoichiometric imbalance among members of the complex, resulting in a deleterious dominant negative phenotype. Finally, ohnologs were observed to be resistant to fluctuations in relative quantities by SSD and the CNV (21, 39), and, consistent with this, they are strongly associated with disease. In particular, Down syndrome caused by trisomy 21 appears to be caused in large part by the deleterious effects of the 1.5-fold increase in dosage of ohnologs on that chromosome (21).In the present study we created three lists of genes from known or potentially pathogenic CNVs, and tested these for inclusion of ohnologs. These comprised CNVs associated with schizophrenia and other neurodevelopmental disorders, de novo CNVs under negative genetic selection identified in an Icelandic sample, which are possibly associated with disorders causing reduced fecundity (5), and CNVs from a comprehensive CNV morbidity map of neurodevelopmental delay (40). The proportion of ohnologs in these pathogenic CNV sets was compared with control copy number polymorphisms of a similar size range, which should not be under negative genetic selection, identified in the general population from two studies (41, 42). In addition, we tested whether the ohnologs mapping to pathogenic CNVs were more likely to encode proteins from multisubunit complexes, or showed enrichment for biological pathways, being thus more likely to represent dosage-balanced genes.     

Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

In mammals, multiple physiological, metabolic, and behavioral processes are subject to circadian rhythms, adapting to changing light in the environment. Here we analyzed circadian rhythms in the fecal microbiota of mice using deep sequencing, and found that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. Disruption of the host circadian clock by deletion of Bmal1, a gene encoding a core molecular clock component, abolished rhythmicity in the fecal microbiota composition in both genders. Bmal1 deletion also induced alterations in bacterial abundances in feces, with differential effects based on sex. Thus, although host behavior, such as time of feeding, is of recognized importance, here we show that sex interacts with the host circadian clock, and they collectively shape the circadian rhythmicity and composition of the fecal microbiota in mice.The composition of intestinal microbiota is influenced by host genetics (1), aging (2), antibiotic exposure (3), lifestyle (4), diet (5), pet ownership (6), and concomitant disease (7, 8). The impact of diet in shaping the composition of the microbiota has been well established in both humans and mice (9, 10). The type of food consumed and also the feeding behavior of the host influence the microbiota. For example, a 24-h fast increases the abundance of Bacteroidetes and reduces that of Firmicutes in mouse cecum, without altering the communal microbial diversity (11). Bacteroidetes are also dominant in the microbiota of the fasted Burmese python, whereas ingestion of a meal shifts the intestinal composition toward Firmicutes (12).The rotation of the earth results in the oscillation of light during the 24-h cycle. Organisms adapted to this cycle by developing a circadian rhythm, an endogenous and entrainable mechanism that times daily events such as feeding, temperature, sleep-wakefulness, hormone secretion, and metabolic homeostasis (13, 14). In mammals, this rhythm is controlled by a master clock that resides in the suprachiasmatic nucleus of the hypothalamus. It responds to the changing light cycle and signals this information to peripheral clocks in most tissues (15). The core mammalian clock is comprised of activators BMAL1 and CLOCK as well as repressors PERIOD (PER) and CRYPTOCHROME (CRY), forming an interlocked regulatory loop (14).Circadian rhythms also exist in fungi and cyanobacteria (16). For example, a pacemaker in cyanobacteria transduces the oscillating daylight signal to regulate gene expression and to time cell division (17, 18). Hence, the synchronization of endogenous circadian rhythms with the environment is crucial for the survival of the bacteria as well as metazoa.Recent studies show that the intestinal microbiota undergo diurnal oscillation under the control of host feeding time, and that ablation of the host molecular clock Per genes causes dysbiosis (19, 20). Here, we report that microbial composition and its oscillation are influenced by the host clock, including the Bmal1-dependent forward limb of the signaling pathway. We also find that rhythmicity is conditioned by the sex of the host, being more pronounced in females than in males.     

From the Cover: Multigeneration analysis reveals the inheritance,specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.Genome engineering tools are important for plant functional genomics research and plant biotechnology. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been successfully used for efficient genome editing in human cell lines, zebrafish, and mouse (1–3) and recently applied to gene modification in plants (4–10). In this system a short RNA molecule guides the associated endonuclease Cas9 to generate double strand breaks (DSBs) in the target genomic DNA, which lead to sequence mutations as a result of error-prone nonhomologous end-joining (NHEJ) DNA damage repair or to gene correction or replacement as a result of homology-dependent recombination (HR) (11). It was shown that engineered CRISPR/Cas caused mutations in target genes or corrections in transgenes in transient expression assays in plant protoplasts and tobacco leaves (10). Importantly, stable expression of the CRISPR/Cas in transgenic Arabidopsis, tobacco, and rice plants led to mutations (mostly indels) in target genes and correction of a transgene (4–9). However, it was not known whether the gene mutations and corrections occurred in somatic cells only or whether some of the mutations and corrections happened in germ-line cells and thus may be heritable. Additionally, it is unclear how specific the CRISPR/Cas is in plants. Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15). Here we show that the CRISPR/Cas-induced transgene correction or mutations in endogenous plant genes and transgenes detected in Arabidopsis T1 plants occurred mostly in somatic cells. However, some of the gene modifications were transmitted through the germ line and were heritable in Arabidopsis T2 and T3 plants following the classic Mendelian model. Mutations caused during DSB repair were predominantly 1 bp insertion and short deletions. Furthermore, our deep sequencing and analysis did not detect any off-targets in multiple CRISPR/Cas transgenic Arabidopsis lines, indicating that the mutagenesis effect of CRISPR/Cas is highly specific in plants.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.