Gene expression changes in male accessory glands during ageing are accompanied by reproductive decline in Drosophila melanogaster.

Senescence is accompanied by loss of reproductive functions. Here, we studied reproductive ageing in Drosophila melanogaster males and asked whether the expected decline in male reproductive success is due to diminished functionality of the male accessory gland (AG). The male AG produces the majority of seminal fluid proteins (SFPs) transferred to the female at mating. SFPs induce female postmating changes and are key to male reproductive success. We measured age-dependent gene expression changes for five representative SFP genes in males from four different age groups ranging from 1 to 6 weeks after eclosion. Simultaneously, we also measured male reproductive success in postmating traits mediated by transfer of these five SFPs. We found a decreased in male SFP gene expression with advancing age and an accompanying decline in male postmating success. Hence, male reproductive senescence is associated with a decline in functionality of the male AG. While overall individual SFP genes decreased in expression, our results point towards the idea that the composition of an ejaculate might change with male age as the rate of change was variable for those five genes.

Koppik M ，Fricke C 《-》

The effect of mating history on male reproductive ageing in Drosophila melanogaster.

Mating bears costs, but how these costs affect the senescence of reproductive traits in males has received relatively little attention. Males of many species show reduced benefits from pre- and post-copulatory reproductive traits during ageing. Senescence of post-copulatory reproductive traits is often linked to a reduction in sperm quantity and quality, but can also be a consequence of changes in seminal fluid proteins that are transferred alongside sperm during mating. Here we investigated how mating history affects male reproductive ageing, especially at the post-copulatory level, using Drosophila melanogaster, a species in which links between seminal fluid proteins and male reproductive traits are well established. Besides a male cohort kept virgin until the start of the experiment we also included a cohort of males kept together with females allowing for ample mating opportunities. With these males we conducted a series of behavioral experiments covering several aspects of male reproductive success with males ranging in age from 4 days to 6 weeks after eclosion. Additionally, we investigated the storage capacity of male accessory glands (AG), the production site of the majority of seminal fluid proteins. We found male reproductive success to decline with increasing male age and, most importantly, males with prior matings showed a reduced performance in pre-copulatory success. However, our data suggest a constant short-term cost of mating rather than an accelerated senescence of pre-copulatory traits. In contrast, senescence of post-copulatory reproductive traits differed between mated and virgin males, hinting at mating costs in males altering the ageing process. We could not find any differences in the capacity of the AG to store seminal fluid proteins, however, our data suggest that old males transfer fewer seminal fluid proteins in a single mating. We conclude that a variety of traits is affected by male reproductive ageing in D. melanogaster with the cost of mating varying in its impact on senescence in these traits.

Koppik M ，Ruhmann H ，Fricke C 《-》

The impact of ageing on male reproductive success in Drosophila melanogaster.

Male reproductive ageing has been mainly explained by a reduction in sperm quality with negative effects on offspring development and quality. In addition to sperm, males transfer seminal fluid proteins (Sfps) at mating; Sfps are important determinants of male reproductive success. Receipt of Sfps leads to female post-mating changes including physiological changes, and affects sperm competition dynamics. Using the fruit fly Drosophila melanogaster we studied ageing males' ability to induce female post-mating responses and determined the consequences of male ageing on their reproductive success. We aged males for up to 7weeks and assayed their ability to: i) gain a mating, ii) induce egg-laying and produce offspring, iii) prevent females from remating and iv) transfer sperm and elicit storage after a single mating. We found that with increasing age, males were less able to induce post-mating responses in their mates; moreover ageing had negative consequences for male success in competitive situations. Our findings indicate that with advancing age male flies transferred less effective ejaculates and that Sfp composition might change over a male's lifetime in quantity and/or quality, significantly affecting his reproductive success.

Ruhmann H ，Koppik M ，Wolfner MF ，Fricke C ... -  《-》

The evolution of seminal fluid gene expression and postmating reproductive isolation in Drosophila.

Seminal fluid protein (Sfp) genes show, in general, a higher rate of sequence divergence than genes from other categories, which is often attributed to forms of postcopulatory sexual selection or sexual conflict. Recently, the relaxation of selective constraints has been proposed as an alternative explanation for the rapid sequence evolution of Sfps and other genes with sex-limited expression. The expression of Sfp genes is a likely target of selection, but the evolution of differences in their expression levels is less understood. Here, we explore both polymorphism and divergence in Sfp gene expression between Drosophila melanogaster and Drosophila simulans, how selection might have influenced their expression, and whether changes in expression might trigger the evolution of reproductive isolating barriers. In our analysis, Sfp genes showed higher divergence, but not higher polymorphism, in expression than the average male reproductive glands gene. Sfp genes with reproductive-tissue-specific expression were enriched for both directional and stabilizing selection, while relaxed selection was the predominant mode of evolution among Sfp genes with any other nonreproductive tissue-specific or nontissue-specific expression. The knockdown of single genes known to affect intraspecific sperm competition, and with patterns of expression divergence and polymorphism suggestive of directional selection, was not enough to break down postmating reproductive isolation barriers between species. Our results identify the expression of male-specific Sfp genes as an enriched target of selection and suggest a complex molecular relationship between postcopulatory sexual selection on a single gene's expression and its effect on the onset of speciation.

Flacchi C ，Capri N ，Civetta A 《-》

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes.

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.

LaFlamme BA ，Ram KR ，Wolfner MF 《PLoS Genetics》