Biology subjects

Carlisle, J. A.

Publications and source records attributed to Carlisle, J. A..

8 recordsLinked to original sources

bioRxiv2026

Female genetic variation controlling timing of mating plug ejection in Drosophila melanogaster

In multiply-mating species, male-female postcopulatory, prezygotic interactions can influence reproductive outcomes. In Drosophila melanogaster, females can bias sperm storage and usage and thereby influence paternity outcomes. One mechanism by which females may regulate paternity contributions from specific males is through modulation of mating plug ejection timing. The D. melanogaster mating plug is composed of seminal fluid proteins, and some female-derived proteins, that coagulate in the female reproductive tract during mating. The mating plug facilitates sperm storage; thus, timing of female mating plug ejection is associated with sperm storage and relative paternity contributions in cases of multiple mating. However, whether there is natural genetic variation among females that shapes mating plug ejection timing, and genes or phenomena that might mediate it are unknown. We examined mating plug ejection in females from 69 lines of the Drosophila Genetic Reference Panel and observed dramatic differences in median plug ejection timing ranging from less than 1 to over 6 hours. We used this variation to perform a genome-wide association study to identify gene candidates associated with this phenotype. Many gene candidates are expressed in the brain and/or function in neurodevelopment. The candidate pool was also enriched for genes expressed in the ovary and functioning in oogenesis, indicating a link between female reproductive physiology and mating plug ejection. Consistent with this interpretation, females without a germline delay mating plug ejection. Our results demonstrate that female mating plug ejection is a physiologically integrated reproductive trait with a genetic basis that can be shaped by selection. Article SummaryThe D. melanogaster mating plug is composed of seminal fluid proteins and some female-derived proteins that coagulate in the female reproductive tract during mating. The mating plug facilitates sperm storage; thus, timing of female mating plug ejection is associated with sperm storage and relative paternity contributions in cases of multiple mating. Using the DGRP, we observed heritable genetic variation in female timing of mating plug ejection and through a GWAS find associated gene candidates. Gene candidates are enriched for neurodevelopment function and oogenesis function. We experimentally validate the connection between female mating plug ejection and the ovary.

genetics↗

bioRxiv2025

Molecular evolution of octopamine receptors in Drosophila

Octopamine (OA), the insect analog of noradrenaline, plays important roles in diverse behavioral and physiological processes, from modulating fight-or-flight behavior to regulating post-mating ovulation. In Drosophila, six OA receptors have been identified: Oamb, Oct2R, Oct{beta}1R, Oct{beta}2R, Oct{beta}3R, and Oct-TyrR, and they have been linked to different behavioral and physiological processes. Here, we investigated the evolutionary characteristics of these receptors across Drosophila species. We found that OA receptors are generally found as single-copy genes. Notably, Oct{beta}2R and Oct{beta}3R exhibit positive selection within the melanogaster group, though in different structural regions from one another. The positively selected sites in Oct{beta}2R are exclusively located in regions important for ligand binding, whereas those in Oct{beta}3R are predominantly found in regions crucial for signal transduction. Interestingly, Oct{beta}2R remains highly conserved outside the melanogaster group, so the detection of positive selection in its ligand-binding related domains within this clade raises the possibility that it has evolved an additional, melanogaster-specific ligand interaction(s), among other potential reasons. These findings highlight the evolutionary flexibility of aminergic signaling and suggest lineage-specific adaptations of OA receptor function in Drosophila, likely shaped by lineage-specific selective pressures. Article summaryOctopamine is a chemical messenger in insects, analogous to noradrenaline in vertebrates, and influences many processes such as fight-or-flight responses, locomotion, and reproduction. In fruit flies and other insects, multiple octopamine receptors detect and respond to this signal. We explored how these receptors have changed over millions of years across various fly species. We found that while some receptors remain similar, others have evolved more rapidly, possibly due to changes in the insects lifestyles, environments, or other biological demands. Understanding these differences can help reveal how insects fine-tune their behaviors and adapt to diverse challenges.

evolutionary biology↗

bioRxiv2025

Correlated Gene Copy Number Changes in a Seminal Fluid Protein Network in Drosophila

Reproductive proteins often diverge rapidly between species, yet network function must be maintained. Shared selective pressures on network members and compensatory changes between members can drive their parallel evolutionary trajectories. Indeed, correlated evolutionary rates of amino acid sequence change have been observed for interacting reproductive proteins. But whether gene copy number changes also correlate has not been widely studied. Here, we investigated copy number variation (CNV) of genes in the Drosophila Sex Peptide Seminal Fluid Protein (Sfp) network. Previous research analyzed CNV of the Sfp Sex Peptide (SP) in Drosophila species. We focus on 9 other Sfps whose function is required to mediate the binding of SP to sperm in D. melanogaster which is required for persistence of female post-mating responses. To exhaustively annotate CNV of genes, we developed a computational pipeline pairing iterative protein queries to genome sequence searches with phylogenetic clustering to resolve homology relationships. We observed that the Sfp networks genes are ancestral to Drosophila and that there were repeated duplications and losses of network members across the genus. We detect statistically significant correlations in gene duplication or loss events among network proteins, and show this can be used to identify new members of the network. We also investigated CNV of female-derived proteins that act downstream of the SP sperm-binding network to modulate SP function, these proteins showed no significant correlation of gene turnover events with SP or its network. Our results provide insight into how evolving reproductive genes tolerate duplication and loss, and how network relationships could constrain reproductive protein evolution. Significance StatementReproductive proteins often diverge rapidly between species, yet network function must be maintained. Shared selective pressures on network members and compensatory changes between members can drive their parallel evolutionary trajectories. We report correlated gene duplication and loss among members of the Drosophila Sex Peptide seminal fluid protein network, suggesting that duplication or loss events may drive corresponding events in other network genes. This work is a natural extension of the idea of evolutionary rate covariation, but instead of scoring rates of substitution it tracks correlated duplication and loss events on the phylogeny. Applied to the Sex Peptide network, the method reveals striking patterns, especially for coordinated loss, and identifies a new network gene that is experimentally confirmed.

evolutionary biology↗

bioRxiv2023

Decoupled evolution of the Sex Peptide gene family and Sex Peptide Receptor in Drosophilidae

Across internally fertilising species, males transfer ejaculate proteins that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 264 species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses in Drosophila melanogaster. We infer that SP first evolved in the Drosophilinae subfamily and has followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa, SP exists largely as a single-copy gene with independent losses in several lineages. Within the Sophophora-Lordiphosa, the SP gene family has repeatedly and independently expanded. Up to seven copies, collectively displaying extensive sequence variation, are present in some species. Despite these changes, SP expression remains restricted to the male reproductive tract. Alongside, we document considerable interspecific variation in the presence and morphology of seminal microcarriers that, despite the critical role SP plays in microcarrier assembly in D. melanogaster, appear to be independent of changes in the presence/absence or sequence of SP. We end by providing evidence that SPs evolution is decoupled from that of its receptor, SPR, in which we detect no evidence of correlated diversifying selection. Collectively, our work describes the divergent evolutionary trajectories that a novel gene has taken following its origin and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor. SignificanceIn insects, seminal fluid proteins (SFPs) induce dramatic changes in female behaviour and physiology. How this degree of male influence evolves remains a central question in sexual selection research. Here, we map the origin and diversification of the posterchild insect SFP, the Drosophila Sex Peptide (SP), across 264 Diptera species. We show that SP first evolved at the base of the subfamily Drosophilinae and followed markedly different evolutionary trajectories in different lineages, including accelerated change in sequence, copy number, and genomic position in the lineage leading to D. melanogaster. By contrast, we find only limited, uncorrelated change in the sequence of its receptor, SPR, arguing against a sexually antagonistic coevolutionary arms race between these loci on macroevolutionary time scales.

evolutionary biology↗

bioRxiv2023

The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract.

Unique patterns of inheritance and selection on Y chromosomes lead to the evolution of specialized gene functions. Yet characterizing the function of genes on Y chromosomes is notoriously difficult. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. WDY mutants produce mature sperm with beating tails that can be transferred to females but fail to enter the female sperm storage organs. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as wild-type sperms and that the mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely cause the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggest that sperm tail beating and propulsion do not always correlate. Furthermore, we find significant differences in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs of WDY that are Y-linked and those that are autosomal. Given that WDY appears to be evolving under positive selection, our results suggest that WDYs functional evolution coincides with its transition from autosomal to Y-linked in Drosophila melanogaster and its most closely related species. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. In contrast to WDY, PRY mutants do swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

genetics↗

bioRxiv2021

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys

In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction. ZP3r, as a member of the RCA cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Sequence divergence paired with paralogous relationships with neighboring genes, has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and many monkey species. ZP3r in both primates that retain ZP3r and rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3rs relative importance to ZP recognition varies across species.

molecular biology↗

bioRxiv2021

Recurrent Duplication and Diversification of Acrosomal Fertilization Proteins in Abalone

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another iimportant mechanism that facilitates the generation of molecular novelty. Following duplication, paralogs may parse ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, consistent with a subfunctionalization model where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.

molecular biology↗

bioRxiv2017

Trait specificity mediated by alternative DNA-binding preferences of a single transcription factor in yeast.

In Saccharomyces cerevisiae, the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the co-factor Tec1. Here, we characterize the in vitro binding preferences of Ste12 to identify a defined spacing and orientation of dimeric sites, one that is common in pheromone-regulated genes. We find that single amino acid changes in the DNA-binding domain of Ste12 can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. Some exceptional Ste12 mutants promote hyperinvasion in a Tec1-independent manner; these fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites that contain one highly degenerate half site. We propose a model for how activation of invasion genes could have evolved with Ste12 alone.

genomics↗