Seminal fluid-mediated fitness effects in the simultaneously hermaphroditic flatworm Macrostomum lignano

Weber M, Giannakara A, Ramm SA. Seminal fluid-mediated fitness effects in the simultaneously hermaphroditic flatworm Macrostomum lignano. Ecology and evolution. 2019;9(24):13889-13901.As a class, seminal fluid proteins are expected to exert strong effects on mating partners due to the selection pressures of sperm competition and sexual conflict. But because of the complexity of this secretion, linking specific proteins to downstream effects on own fitness-via manipulating the reproductive behavior, physiology, and ultimately the sperm utilization of mating partners-is not straightforward. Here, we adopted a systematic gene knockdown approach to screen for seminal fluid-mediated fitness effects in the simultaneously hermaphroditic flatworm Macrostomum lignano. We focused on 18 transcripts in M. lignano seminal fluid, testing how their RNA interference-induced knockdown impacted on three aspects of donor (male) reproductive success: (a) fertility (offspring production of the partner); (b) defensive sperm competitive ability, P-1; and (c) offensive sperm competitive ability, P-2. In general, the knockdown of most individual transcripts appeared to have only a minor impact on male reproductive success, though we found evidence that the knockdown of up to five different transcripts impacted on fertility; the knockdown of two other transcripts resulted in reduced P-2; and knockdown of a further transcript actually increased P-2. We thus identify a number of candidate seminal fluid transcripts that appear to modulate offspring production and sperm competitiveness in M. lignano. That only a minority of transcripts exhibit such a pattern likely reflects both the difficulty of accurately estimating sperm competitiveness and the functional redundancy of seminal fluid

Effects of two seminal fluid transcripts on post‐mating behavior in the simultaneously hermaphroditic flatworm Macrostomum lignano

Weber M, Patlar B, Ramm SA. Effects of two seminal fluid transcripts on post‐mating behavior in the simultaneously hermaphroditic flatworm Macrostomum lignano. Journal of Evolutionary Biology. 2020;22(5): jeb.13606.The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post-mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partners' (sperm recipient's) post-copulatory behavior to prevent or delay re-mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioral data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs - Mlig-pro46 and Mlig-pro63 - linked to both mating frequency and 'suck' frequency, a distinctive behavior, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference-induced knockdown to ask how the loss of Mlig-pro46 and Mlig-pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knock-down treatments impacted strongly on the mating frequency or sperm competitive ability, but knock-down of Mlig-pro63 resulted in a significantly decreased 'suck' propensity of mating partners. This suggests that Mlig-pro63 may normally act as a cue in the ejaculate to trigger recipient suck behavior, and - given that other proteins in the ejaculate have the opposite effect - could be one component of an on-going arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig-pro46 and Mlig-pro63 from a donor perspective remains enigmatic. This article is protected by copyright. All rights reserved

Sperm competition and the evolution of spermatogenesis

Spermatogenesis is a long and complex process that, despite the shared overall goal of producing the male gamete, displays striking amounts of interspecific diversity. In this review, we argue that sperm competition has been an important selection pressure acting on multiple aspects of spermatogenesis, causing variation in the number and morphology of sperm produced, and in the molecular and cellular processes by which this happens. We begin by reviewing the basic biology of spermatogenesis in some of the main animal model systems to illustrate this diversity, and then ask to what extent this variation arises from the evolutionary forces acting on spermatogenesis, most notably sperm competition. We explore five specific aspects of spermatogenesis from an evolutionary perspective, namely: (i) interspecific diversity in the number and morphology of sperm produced; (ii) the testicular organizations and stem cell systems used to produce them; (iii) the large number and high evolutionary rate of genes underpinning spermatogenesis; (iv) the repression of transcription during spermiogenesis and its link to the potential for haploid selection; and (v) the phenomenon of selection acting at the level of the germline. Overall we conclude that adopting an evolutionary perspective can shed light on many otherwise opaque features of spermatogenesis, and help to explain the diversity of ways in which males of different species perform this fundamentally important proces

Sperm competition risk drives plasticity in seminal fluid composition

Background Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment. Results Our quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid – i.e. seminal fluid proteome composition – differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition – SVS 6, SVS 5 and CEACAM 10 – were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities. Conclusions Our study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition

The baculum affects paternity success of first but not second males in house mouse sperm competition

The vast variation observed in genital morphology is a longstanding puzzle in evolutionary biology. Studies showing that the morphology of the mammalian baculum (penis bone) can covary with a male’s paternity success indicate a potential impact of baculum morphology on male fitness, likely through influencing sperm competition outcomes. We therefore measured the size (measurements of length and width) and shape (geometric morphometric measurements) of the bacula of male house mice used in previously published sperm competition experiments, in which two males mated successively with the same female in staged matings. This enabled us to correlate baculum morphology with sperm competition success, incorporating potential explanatory variables related to copulatory plugs, male mating behavior and a selfish genetic element that influences sperm motility. We found that a wider baculum shaft increased a male’s paternity share when mating first, but not when mating second with a multiply-mating female. Geometric morphometric shape measurements were not clearly associated with fertilization success for either male. We found limited evidence that the effect of baculum morphology on male fertilization success was altered by experimental removal of the copulatory plug. Furthermore, neither genetic differences in sperm motility, nor covariation with male mating behavior mediated the effect of baculum morphology on male fertilization success. Taken together with previous findings, the mating-order effects we found here suggest that baculum-mediated stimulation by the first male might be particularly important for fertilization

Impact of low sperm competition on male reproductive trait allometries in a bush-cricket

Winkler L, Kirch LM, Reinhold K, Ramm SA. Impact of low sperm competition on male reproductive trait allometries in a bush-cricket. BMC Evolutionary Biology. 2019;19(1): 185.Background Studying reproductive trait allometries can help to understand optimal male investment strategies under sexual selection. In promiscuous mating systems, studies across several taxa suggest that testes allometry is usually positive, presumably due to strong selection on sperm numbers through intense sperm competition. Here, we investigated testes allometry in a bush-cricket species, Metaplastes ornatus, in which females mate promiscuously, but where sperm removal behaviour by males likely drastically reduces realised sperm competition level. Results As hypothesised, we found evidence for negative testes allometry and hence a fundamentally different male investment strategy compared to species under intense sperm competition. In addition, the mean relative testes size of M. ornatus was small compared to other species of bush-crickets. Surprisingly, the spermatophore gland, a potential alternative trait that males could invest in instead of testes, also did not show positive allometry, but was approximately isometric. We further observed the expected pattern of negative allometry for the male morphological structure responsible for sperm removal in this species, the subgenital plate, supporting the one-size-fits-all hypothesis for intromittent genitalia. Conclusion Our findings suggest that the evolution of sperm removal behaviour in M. ornatus was a key adaptation for avoiding sperm competition, with important consequences for reproductive trait allometries. Nevertheless, they also imply that it does not pay for larger males to invest disproportionately in nuptial gift production in this species

The free-living flatworm Macrostomum lignano

Wudarski J, Egger B, Ramm SA, et al. The free-living flatworm Macrostomum lignano. EvoDevo. 2020;11(1): 5

Sperm competition-induced plasticity in the speed of spermatogenesis

Background: Sperm competition between rival ejaculates over the fertilization of ova typically selects for the production of large numbers of sperm. An obvious way to increase sperm production is to increase testis size, and most empirical work has focussed on this parameter. Adaptive plasticity in sperm production rate could also arise due to variation in the speed with which each spermatozoon is produced, but whether animals can respond to relevant environmental conditions by modulating the kinetics of spermatogenesis in this way has not been experimentally investigated. Results: Here we demonstrate that the simultaneously hermaphroditic flatworm Macrostomum lignano exhibits substantial plasticity in the speed of spermatogenesis, depending on the social context: worms raised under higher levels of sperm competition produce sperm faster. Conclusions: Our findings overturn the prevailing view that the speed of spermatogenesis is a static property of a genotype, and demonstrate the profound impact that social environmental conditions can exert upon a key developmental process. We thus identify, to our knowledge, a novel mechanism through which sperm production rate is maximised under sperm competition

A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm

Weber M, Wunderer J, Lengerer B, et al. A targeted in situ hybridization screen identifies putative seminal fluid proteins in a simultaneously hermaphroditic flatworm. BMC Evolutionary Biology. 2018;18(1): 81.Background Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner’s behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano. Results Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation. Conclusions Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs

Radiation therapy attenuates lymphatic vessel repair by reducing VEGFR-3 signalling

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood.Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema.Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury.Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema