Structural complexity and molecular heterogeneity of a butterfly ejaculate reflect a complex history of selection

Abstract

Male ejaculates are often structurally complex, and this complexity is likely to influence key reproductive interactions between males and females. However, despite its potential evolutionary significance, the molecular underpinnings of ejaculate structural complexity have received little empirical attention. To address this knowledge gap, we sought to understand the biochemical and functional properties of the structurally complex ejaculates of butterflies. Males in this species produce large ejaculates called spermatophores composed of an outer envelope, an inner matrix, and a bolus of sperm. Females are thought to benefit from the nutrition contained in the soluble inner matrix through increases in longevity and fecundity. However, the indigestible outer envelope of the spermatophore delays female remating, allowing males to monopolize paternity for longer. Here, we show that these two nonsperm-containing spermatophore regions, the inner matrix and the outer envelope, differ in their protein composition and functional properties. We also reveal how these divergent protein mixtures are separately stored in the male reproductive tract and sequentially transferred to the female reproductive tract during spermatophore assembly. Intriguingly, we discovered large quantities of female-derived proteases in both spermatophore regions shortly after mating, which may contribute to spermatophore digestion and hence, female control over remating rate. Finally, we report evidence of past selection on these spermatophore proteins and female proteases, indicating a complex evolutionary history. Our findings illustrate how structural complexity of ejaculates may allow functionally and/or spatially associated suites of proteins to respond rapidly to divergent selective pressures, such as sexual conflict or reproductive cooperation.

Microevolutionary selection dynamics acting on immune genes of the green‐veined white butterfly, Pieris napi

“…The Pieris napi genome assembly (7,829 scaffolds, N50 = 300,294) was reported to contain a conserved single‐copy ortholog content of 78% using CEGMA (Meslin et al., ). Here, this was further assessed by quantifying the content of complete BUSCO arthropod genes, which was 74% (Supporting information Table ), similar to other recently published Lepidopteran genomes, which ranged from 67.7% to 99.1% (Kanost et al., ; Supporting information Table ).…”

“…The P. napi and P. rapae genome sequences used for the MK test were obtained from Meslin et al. (). The P. brassicae genome was constructed using a Pool‐seq library from the thoraxes of 24 Spanish butterflies (41.818225°N, 2.302625°E; 12 males and 12 females).…”

“…Here, we use a draft genome of Pieris napi, previously only used as a database for identifying <100 genes for a comparative analysis (Meslin et al, 2017). As this genome lacks an annotation, we generated a draft annotation using SPALN v2.1.2 (Gotoh, 2008;Iwata & Gotoh, 2012), which is a high-performance protein to genome alignment program that is exon boundary aware, and the Danaus plexippus protein set DPOGS2 (Zhan & Reppert, 2013).…”

BMP-signalling inhibition inDrosophilasecondary cells remodels the seminal proteome, and self and rival ejaculate functions

“…It has been suggested that by sequestering SFPs in different cells or glands males are afforded control over their release, and consequently, spatiotemporal control over their interactions with sperm, the female reproductive tract, or with other SFPs (16). Additionally, functional diversification of tissues and cell-types may be required to build specialised parts of the ejaculate, such as mating plugs (17). In either case, activities may be carried out independently between cell-types and tissues or there may be cross-talk between them that coordinates global seminal fluid composition.…”

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones

“…Such sexual selection can also influence molecular traits and has resulted in many reproductive proteins that evolve at extraordinary rates [12,13]. For example, in the marine gastropod abalone, males produce an extraordinary number of sperm that overexpress the rapidly evolving protein lysin (up to ~1 g lysin per male abalone) that facilitates species-specific fertilization [14][15][16]; in fruit flies, males accessory glands synthesize complex mixtures of rapidly evolving seminal fluid proteins that restrict female re-mating by reducing her viability and survival [17,18]; similarly, in Pieris butterflies, males produce enormous spermatophores (~13% of their body mass) that are encased in a nearly indestructible protein shell that slows spermatophore clearance and prevents female re-mating [19]. In these examples, however, the molecular mechanisms underlying the regulated expression of these unusual reproductive proteins are not fully understood.…”