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

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.

Evolution of Secondary Cell Number and Position In the Drosophila Accessory Gland

“…2006) or the expression of different homologues in the AG of different species (Ahmed-Braima et al 2017; Cridland et al . 2020, 2022; Hurtado et al . 2022).…”

“…Recent RNA-seq analysis of the SC has revealed that its transcriptome is distinct from that of the main cells and also evolves in a distinct manner (Immarigeon et al . 2021, Majane et al . 2022).…”

“…Sfp protein sequences often evolve very quickly (Coulthart and Singh 1988), sometimes under the influence of directional selection (Begun et al 2000;Swanson et al 2001;Wagstaff and Begun 2005a;Haerty et al 2007). Expression levels of AG-biased genes may vary widely even between closely related species (Begun and Lindfors 2005, Ahmed-Braima et al 2017, Garlovsky et al 2020, and the AG transcriptome exhibits rapid turnover due to gene presence/absence variation 2005b, Mueller et al 2005;Begun et al 2006) or the expression of different homologues in the AG of different species (Ahmed-Braima et al 2017;Cridland et al 2020Cridland et al , 2022Hurtado et al 2022).…”

“…Secondary cells (SC) play a role in the secretion of extracellular vesicles (i.e., exosomes), which bind to sperm, interact with the female reproductive tract epithelium, and play a role in the long-term mating response of females (Corrigan et al 2014). Recent RNA-seq analysis of the SC has revealed that its transcriptome is distinct from that of the main cells and also evolves in a distinct manner (Immarigeon et al 2021, Majane et al 2022. Given that male reproductive proteins and transcriptomes evolve quickly and that mating systems vary dramatically across Drosophila (Markow 1996), whether most basic conclusions about the AG as described in the D. melanogaster model apply generally to Drosophila is an open question.…”