The cactophilic D. mojavensis and D. arizonae are promiscuous, mating multiple times a day, even heterospecifically in lab settings (Diaz et al unpublished data). Yet these species have remained isolated for ~ 0.5My, suggesting the presence of multiple reproductive barriers preventing introgressive hybridization [35, 66, 67]. PMPZ isolation has been confirmed for crosses involving D. mojavensis females [21], where the reaction mass is more evident and molecular interactions in the female lower reproductive tract were found altered by the heterospecific ejaculate [8]. Here, we demonstrate that copulation induces substantial transcriptional changes in head tissues of females that are substantially perturbed when mating with a heterospecific male in both species. These changes compromise functional pathways important for the female postcopulatory physiology and behavior that normally would be expressed in conspecifically-mated females, some of these evolving rapidly, which might have implications for the extent of sexual conflict [68].
Our results indicate that mating induces not only gene expression changes in female heads soon after mating, but also that a great part of this response is controlled by alternative splicing. The number of genes responding through AS often exceeded that of DE genes, but both mechanisms appear to be involved in the female postcopulatory response. We examined AS patterns caused by multiple mechanisms, including intron retention – IR [18], when comparing mated vs virgin females. Differential usage of examined gene features showed substantial consequences for the postmating response that were not reflected in gene expression of head transcriptomes. The role of AS in the postmating response has not been previously evaluated, but is consistent with the complexity of epistatic interactions related to sexual traits [34]. Interestingly, in this study, genes experiencing DE or AS appear to be almost mutually exclusive (less than 5% overlap). Consequently, the female postmating response seems to target different functions through each of these mechanisms. DE genes are mainly linked to pathways of proteolysis and nutrient homeostasis, while AS genes are more related to those involved in photoreception and muscle assembly changes.
IR is a particular case of AS that, although has been associated with some active functional changes, is most likely linked to gene regulation resulting in degradation of the mRNA by the nonsense-mediated mRNA decay – NMD pathway [20, 69]. We demonstrate that IR is not only differentially regulated between con- and heterospecific matings but seems to be an active mechanism of gene regulation as IR rates increased for down regulated genes but decreased for up-regulated genes.
The transcriptional response to mating has been studied in a few insect species [9–16], showing biologically meaningful pathways common to the postmating response across different species. In fact, some of the mating-activated genes that we found in female heads are associated with functional pathways previously reported in different tissues and species. Proteolytic pathways for example, are within the most common and strongly activated genes that are part of the female response, found in both reproductive tissues and whole female bodies [10, 38, 70]. However, this is a complex reproductive response, given that a great part of the male ejaculate is also composed of a diverse cocktail of both proteases and their inhibitors [2].
Increasing interest in SFP have revealed a great array of proteases with diverse functions in both testis and the female postmating response [4, 12, 71]. Most of these are related to earlier postmating processes and molecular interactions occurring in the female tract (e.g. sperm storage and SFP cleavage) [71–73], but it is unclear whether the proteases or inhibitors expressed by the female are involved in the same functions. However, the lasting effects, even several days after mating, and the fact that they have been detected in several species, from insects to mammals [4, 73], suggest that these proteolytic cascades are involved in multiple functions of the whole organism mating response. For example, the expression of proteolytic cascades we observed in heads of mated females does not seem connected to sperm-related functions that occur in the female reproductive tract. One possibility is that some of these cascades are involved in protein degradation for amino acid related pathways and nutrient homeostasis, a hypothesis supported by our functional analysis. We identified two functional categories associated with Carnitine biosynthesis. This particular amino acid is known to start with the degradation of proteins containing N-methylated lysine by proteolytic cascades [74], with a major role in energy homeostasis in the nervous system [39, 40].
We detected several biosynthetic pathways associated with the production of specific amino acids important for nutrient homeostasis such as carnitine, threonine, and leucine. Although such pathways are not directly classified as behavioral or reproductive, some of their metabolic functions have been previously detected in conspecific experiments in D. melanogaster [28, 38]. These networks regulate energy balance [39, 40] and nutritional uptake in brain tissues [41], and have been directly linked to several postcopulatory behaviors, including circadian rhythms [42, 43], nutrient sensing, exploratory behavior for specific nutrient source, consumption and posterior oviposition [26, 27, 75, 76]. Consequently, mated D. melanogaster females experience a major switch in their diet following copulation [77], consuming more amino acids during the dark phase [26]. The mechanisms of this behavioral switch have been linked to neuronal signaling triggered by SP-SPR dynamics in D. melanogaster [78–80].
Transcriptional dynamics detected in female heads of both species were substantially perturbed by heterospecific matings. Thus, changes of regulatory networks related to nutrient homeostasis in brain tissues were characteristic of conspecific dynamics in D. mojavensis, while changes detected in D. arizonae females were dominated by proteolytic pathways. The biological function of genes, and the magnitude of their expression were perturbed following copulation with a heterospecific male. Heterospecifically-mated D. mojavensis were unable to activate nutrient-homeostasis- (DE genes) and photoreception-related (AS genes) pathways and activated proteolytic and some muscle assembly genes instead. In contrast, heterospecifically-mated D. arizonae females showed no enriched pathways for DE genes, suggesting strong functional perturbation. These results, suggest that interactions between seminal fluid components and the female tract induce distant epistatic effects in tissues of the female head soon after mating [28, 38]. The mode of signaling for these changes in insects is still an ongoing investigation. Given the distance and heterogeneity of the tissues involved, changes induced in the female head are assumed to be the product of altered interaction networks, influencing the physiology of other tissues [29, 81]. This is more an indirect effect of neurons associated with internal reproductive tissues, which may produce signaling molecules that influence gene expression at distant sites in the fly body [82]. However, evidence from D. melanogaster suggests that seminal fluid components like the SP rapidly circulate in the female’s hemolymph and has been found associated with brain tissues [32]. Furthermore, immediate post-copulatory transcriptional responses in the nervous system could also be due to social interactions, as has been shown in D. melanogaster with males that court but fail to copulate [83]. Further research is needed to disentangle the underlying mechanisms causing the activation of genes outside of the female reproductive tract and their involvement in the post-copulatory response.
The male side of mating has been long been investigated in several species and it is well known that male reproduction genes, and particularly seminal fluid proteins – SFP evolve rapidly [34]. Although the female genes have only been investigated in a few species, results in D. mojavensis and D. virilis female’s reproductive tracts [66, 70, 84] indicated that these genes not always evolve as rapid as the male genes. Here, we found that DE genes in female heads are evolving rapidly in D. arizonae, but not in D. mojavensis. Conspecific genes detected in D. arizonae are related to different proteolytic pathways and showed evolutionary rates even higher than SFP genes previously detected in these species [37]. These pathways were not enriched in D. mojavensis females, but some increased when mated with D. arizonae males. AS genes on the other hand exhibited evolutionary rates even lower than the genome background. These genes were linked to very specific functions of highly conserved genes. Therefore, this could be due to the conservation of those specific gene functions in these species, but more likely seems to be reflecting a general trend in the molecular evolution of AS genes [85]. To date, the role of AS in the female postmating response has not previously evaluated in insect species, nor the relationship between AS and molecular evolution in Drosophila. However, this same result was also previously found when investigating strictly AS genes across the mouse and human genomes [85], suggesting that the level of alternative splicing and molecular evolution at the sequence level are negatively correlated. Constitutive exons of alternative isoforms tend to evolve faster than newly alternatively spliced exons [85, 86]. It is unclear how these heterogenic patterns would affect gene-wide molecular evolution or specific gene families in insects, but highly constrained exons could decrease molecular evolution at the gene level. These genes are also presumed more pleiotropic as alternative isoforms can evolve without major changes in sequence through functional specialization of different exon arrays.