Sperm competition favours intermediate sperm size in a hermaphrodite1.

Sperm competition is a potent mechanism of postcopulatory sexual selection that has been found to shape reproductive morphologies and behaviours in promiscuous animals. Especially sperm size has been argued to evolve in response to sperm competition through its effect on sperm longevity, sperm motility, the ability to displace competing sperm, and ultimately fertilization success. Additionally, sperm size has been observed to co-evolve with female reproductive morphology. Theoretical work predicts that sperm competition may select for longer sperm but may also favour shorter sperm if sperm size trades-off with number. In this study, we studied the relationship between sperm size and postmating success in the free-living flatworm, Macrostomum lignano. Specifically, we used inbred isolines of M. lignano that varied in sperm size to investigate how sperm size translated into the ability of worms to transfer and deposit sperm in a mating partner. Our results revealed a hump-shaped relationship with individuals producing sperm of intermediate size having the highest sperm competitiveness. This finding broadens our understanding of the evolution of sperm morphology by providing empirical support for stabilizing selection on sperm size under sperm competition.

Effects of two seminal fluid transcripts on post-mating behaviour in the simultaneously hermaphroditic flatworm Macrostomum lignano.

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 partner's (sperm recipient's) post-copulatory behaviour 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 behavioural 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 behaviour, 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 knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown 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 behaviour and-given that other proteins in the ejaculate have the opposite effect-could be one component of an ongoing 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.

Flatworm mucus as the base of a food web.

BACKGROUND: By altering their habitats, engineering species can improve their own fitness. However, the effect of this strategy on the fitness of coexisting species or on the structure of the respective food web is poorly understood. In this study, bacteria and bacterivorous nematodes with short (Caenorhabditis elegans) and long (Plectus acuminatus) life cycles were exposed to the mucus secreted by the freshwater flatworm Polycelis tenuis. The growth, reproduction, and feeding preferences of the nematodes in the presence/absence of the mucus were then determined. In addition, confocal laser scanning microscopy (CLSM) was used to examine the structural footprint of the mucus and the mucus colonization dynamics of bacteria and protozoans. RESULTS: Mucus exposure resulted in a greater reproductive output in P. acuminatus than in C. elegans. In a cafeteria experiment, both nematode species were attracted by bacteria-rich patches and were not deterred by mucus. CLSM showed that the flatworms spread a layer of polysaccharide-rich mucus ca. 15 µm thick from their tails. Subsequent colonization of the mucus by bacteria and protozoans resulted in an architecture that progressively resembled a complex biofilm. The presence of protozoans reduced nematode reproduction, presumably due to competition for their bacterial food supply. CONCLUSION: Animal secretions such as mucus may have broader, community-level consequences and contribute to fueling microbial food webs.

Bigger testes increase paternity in a simultaneous hermaphrodite, independently of the sperm competition level.

Hermaphroditic animals face the fundamental evolutionary optimization problem of allocating their resources to their male vs. female reproductive function (e.g. testes and sperm vs. ovaries and eggs), and this optimal sex allocation can be affected by both pre- and post-copulatory sexual selection. For example, local sperm competition (LSC) - the competition between related sperm for the fertilization of a partner's ova - occurs in small mating groups and can favour a female-biased sex allocation, because, under LSC, investment into sperm production is predicted to show diminishing fitness returns. Here, we test whether higher testis investment increases an individual's paternity success under sperm competition, and whether the strength of this effect diminishes when LSC is stronger, as predicted by sex allocation theory. We created two subsets of individuals of the simultaneously hermaphroditic flatworm Macrostomum lignano - by sampling worms from either the highest or lowest quartile of the testis investment distribution - and estimated their paternity success in group sizes of either three (strong LSC) or eight individuals (weak LSC). Specifically, using transgenic focal individuals expressing a dominant green-fluorescent protein marker, we showed that worms with high testis investment sired 22% more offspring relative to those with low investment, corroborating previous findings in M. lignano and other species. However, the strength of this effect was not significantly modulated by the experienced group size, contrasting theoretical expectations of more strongly diminishing fitness returns under strong LSC. We discuss the possible implications for the evolutionary maintenance of hermaphroditism in M. lignano.

Australian spiny mountain crayfish and their temnocephalan ectosymbionts: an ancient association on the edge of coextinction?

Australian spiny mountain crayfish (Euastacus, Parastacidae) and their ecotosymbiotic temnocephalan flatworms (Temnocephalida, Platyhelminthes) may have co-occurred and interacted through deep time, during a period of major environmental change. Therefore, reconstructing the history of their association is of evolutionary, ecological, and conservation significance. Here, time-calibrated Bayesian phylogenies of Euastacus species and their temnocephalans (Temnohaswellia and Temnosewellia) indicate near-synchronous diversifications from the Cretaceous. Statistically significant cophylogeny correlations between associated clades suggest linked evolutionary histories. However, there is a stronger signal of codivergence and greater host specificity in Temnosewellia, which co-occurs with Euastacus across its range. Phylogeography and analyses of evolutionary distinctiveness (ED) suggest that regional differences in the impact of climate warming and drying had major effects both on crayfish and associated temnocephalans. In particular, Euastacus and Temnosewellia show strong latitudinal gradients in ED and, conversely, in geographical range size, with the most distinctive, northern lineages facing the greatest risk of extinction. Therefore, environmental change has, in some cases, strengthened ecological and evolutionary associations, leaving host-specific temnocephalans vulnerable to coextinction with endangered hosts. Consequently, the extinction of all Euastacus species currently endangered (75%) predicts coextinction of approximately 60% of the studied temnocephalans, with greatest loss of the most evolutionarily distinctive lineages.

Hypodermic self-insemination as a reproductive assurance strategy.

Self-fertilization occurs in a broad range of hermaphroditic plants and animals, and is often thought to evolve as a reproductive assurance strategy under ecological conditions that disfavour or prevent outcrossing. Nevertheless,selfing ability is far from ubiquitous among hermaphrodites, and may be constrained in taxa where the male and female gametes of the same individual cannot easily meet. Here, we report an extraordinary selfing mechanism in one such species, the free-living flatworm Macrostomum hystrix. To test the hypothesis that adaptations to hypodermic insemination of the mating partner under outcrossing also facilitate selfing, we experimentally manipulated the social environment of these transparent flatworms and then observed the spatial distribution of received sperm in vivo. We find that this distribution differs radically between conditions allowing or preventing outcrossing, implying that isolated individuals use their needle-like stylet (male copulatory organ) to inject own sperm into their anterior body region, including into their own head, from where they then apparently migrate to the site of (self-)fertilization. Conferring the ability to self could thus be an additional consequence of hypodermic insemination, a widespread fertilization mode that is especially prevalent among simultaneously hermaphroditic animals and probably evolves due to sexual conflict over the transfer and subsequent fate of sperm.

[ULTRASTRUCTURE OF PARENCHYMA IN THE SYNCYTIAL DIGESTIVE SYSTEM IN TURBELLARIA Convoluta convoluta (Acoela].

The paper presents data on the ultrastructure of parenchyma that is involved in the digestion in turbellaria Convoluta convoluta (n = 15). Unusual connections between the nuclear envelope, endoplasmic reticulum and plasma membrane of parenchymal cells were found for the first time, which may indicate the origin of these cell structures. The double trophic role of zooxanthellae in the organism of Convoluta is described.

The Abyssal Parasitic Flatworm Fecampia cf. abyssicola: New Records, Anatomy, and Molecular Phylogeny, with a Discussion on Its Systematic Position.

AbstractThe order Fecampiida, a group of parasitic turbellarians, has been poorly studied in terms of its species diversity, morphology, and ecology. Fecampiida is positioned within the monophyletic clade Adiaphanida, along with Tricladida and Prolecithophora, but their phylogenetic relationships are not well understood. Although the nervous and muscular systems of only two species in Fecampiida have been studied, recent research inferred morphological similarities between Fecampiida and Prolecithophora. In this study, we collected fecampiid cocoons and juveniles at depths of 1861-4438 m in Japanese waters. We identified the species on the basis of swimming juvenile specimens and by using histological and molecular methods, while we also examined its musculature and nervous system. Our study revealed a more complex nervous system than previously reported, with dorsal, lateral, and ventral pairs of longitudinal nerve cords connected through an anterior neuropile and posterior transverse commissures. While the nervous and muscular morphology suggested similarities with Prolecithophora, our phylogenetic analysis did not support a close relationship between Fecampiida and Prolecithophora.

Proliferative response of the stem cell system during regeneration of the rostrum in Macrostomum lignano (Platyhelminthes).

Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a. Altogether, our data present new insights into the cellular response of the neoblast system upon amputation, clearly demonstrating important differences from the situation known to occur during regeneration of the tail plate. Furthermore, the presence of proliferating cells in the region anterior to the eyes shows a clear alteration in stem cell regulation during regeneration.

[Symbionts of Mytilus edulis in the littoral and sublittoral zones of the Kandalaksha and Onega Gulfs of the White Sea].

Composition of the fauna of organisms associated with Mytilus edulis in the Kandalaksha and Onega Gulfs of the White Sea has been examined. The following 8 symbiotic species were revealed: Choricystis sp. (Chloro[hyceae), Peniculistoma mytili, Ancistrum mytili (Ciliata, Oligohymenophorea), Urastoma cyprinae, Paravortex sp. (Platyhelminthes, Rhabditophora), and metacercaria of Cercaria parvicaudata, Himasthla sp., and Gymnophallus sp. (Platyhelminthes, Trematoda). Besides, different free-living organisms were found in the mantle cavity; 6 species of nematodes, the planktonic copepod Microsetella norvegica, undetermined copepods, isopod Jaera sp., sea mites of the family Halacaridae, and chironomid larvae. Parameters of infestation and places of localization in the host are given for each group of organisms. Some regularities in the horizontal and vertical distribution of organisms associated with M. edulis are noted.

Making heads from tails: development of a reversed anterior-posterior axis during budding in an acoel.

The anterior-posterior axis is a key feature of the bilaterian body plan. Although axis specification during embryogenesis has been studied extensively, virtually nothing is known about how this axis can be established post-embryonically, as occurs in budding animals. We investigated bud formation in the acoel Convolutriloba retrogemma, which reproduces by a remarkable process involving the formation of animals with linked but completely opposite body axes. Reverse axes are established anew during each round of budding and manifestations of the bud's new axis develop gradually, with regionalization of axial patterning genes (Hox and otx) and the establishment of organized musculature occurring secondarily, after bud initiation. A swath of tissue at the parent-bud boundary has no regenerative potential and appears devoid of inherent axial polarity. GSK-3 inhibitor trials suggest that Wnt/beta-catenin or Hedgehog signalling may mediate the establishment of this unpolarized zone. Formation of unpolarized tissue may provide a buffer between opposing polarity cues and be a general mechanism by which budding animals establish and maintain linked body axes. In addition to elucidating the developmental basis of budding in a bilaterian, this study provides insight into convergence in animal budding mechanisms, redeployment of embryonic gene expression during budding, and Hox gene evolution.

SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

Predator functional response changed by induced defenses in prey.

Functional responses play a central role in the nature and stability of predator-prey population dynamics. Here we investigate how induced defenses affect predator functional responses. In experimental communities, prey (Paramecium) expressed two previously undocumented inducible defenses--a speed reduction and a width increase--in response to nonlethal exposure to predatory Stenostomum. Nonlethal exposure also changed the shape of the predator's functional response from Type II to Type III, consistent with changes in the density dependence of attack rates. Handling times were also affected by prey defenses, increasing at least sixfold. These changes show that induced changes in prey have a real defensive function. At low prey densities, induction led to lower attack success; at high prey densities, attack rates were actually higher for induced prey. However, induction increased handling times sufficiently that consumption rates of defended prey were lower than those of undefended prey. Modification of attack rate and handling time has important potential consequences for population dynamics; Type III functional responses can increase the stability of population dynamics and persistence because predation on small populations is low, allowing a relict population to survive. Simulations of a predator-prey population dynamic model revealed the stabilizing potential of the Type III response.

Potential of Macrostomum lignano to recover from gamma-ray irradiation.

Stem cells are the only proliferating cells in flatworms and can be eliminated by irradiation with no damage to differentiated cells. We investigated the effect of fractionated irradiation schemes on Macrostomum lignano, namely, on survival, gene expression, morphology and regeneration. Proliferating cells were almost undetectable during the first week post-treatment. Cell proliferation and gene expression were restored within 1 month in a dose-dependent manner following exposure to up to 150 Gy irradiation. During recovery, stem cells did not cross the midline but were restricted within lateral compartments. An accumulated dose of 210 Gy resulted in a lethal phenotype. Our findings demonstrate that M. lignano represents a suitable model system for elucidating the effect of irradiation on the stem cell system in flatworms and for improving our understanding of the recovery potential of severely damaged stem-cell systems.

Stronger inducible defences enhance persistence of intraguild prey.

1. Intraguild predation is widespread in nature despite its potentially destabilizing effect on food web dynamics. 2. Anti-predator inducible defences affect both birth and death rates of populations and have the potential to substantially modify food web dynamics and possibly increase persistence of intraguild prey. 3. In a chemostat experiment, we investigated the long-term effects of inducible defences on the dynamics of aquatic microbial food webs consisting of an intraguild predator, intraguild prey, and a basal resource. We controlled environmental conditions and selected strains of intraguild prey that varied in the strength of expressed inducible defences. 4. We found that intraguild prey with a stronger tendency to induce an anti-predator morphology persist for significantly longer periods of time. In addition, model selection analysis implied that flexibility in defensive phenotype (inducibility itself) is most likely the factor responsible for the enhanced persistence. 5. As patterns at the community level often emerge as a result of the life-history traits of individuals, we propose that inducible defences increase the persistence of populations and may contribute to the widespread occurrence of theoretically unstable intraguild predation systems in nature.

Bryoplana xerophila n. g. n. sp., a new limnoterrestrial microturbellarian (Platyhelminthes, Typhloplanidae, Protoplanellinae) from epilithic mosses, with notes on its ecology.

Bryoplana xerophila, a new genus and species of limnoterrestrial protoplanelline platyhelminth, was found in moss and soil covering a concrete wall in northern Alabama, USA. Bryoplana xerophila is the first taxon of limnoterrestrial Protoplanellinae recorded from North America and is one of the few rhabdocoels known from dry habitats. It is unique within Protoplanellinae in lacking rhabdites, having a pharynx rosulatus in the frontal half of the body, and lacking sclerotized parts in the male system. Notes on encystment, reproduction and feeding behavior are given. An updated identification key to all known genera of Protoplanellinae is presented.

[Peculiarities of ultrastructure of excretory system in Bothrioplana semperi (Platyhelminthes, Turbellaria)].

Ultrastructural study of morphology of cirtocytes and excretory channels was performed in the free living turbellaria Bothrioplana semperi (Turbellaria, Seriata). It has been shown that cirtocytes of this species are formed by two cells--the terminal and the proximal cells of the channel. The fan is composed of two rod rows. The external row goes out from the terminal cell, the internal one is a derivate of the channel proximal cell. Inside each rod of the external row there runs a bundle of microfilaments; it originates in the cytoplasm of the channel proximal cell distal to bases of the external rods. On the internal rod membranes there are noted small electrondense granules disposed separately or fused in the solid layer continuing into a dense "membrane" connecting rods of the external and internal rows. Rare internal leptotrichiae go out from the cirtocyte cavity bottom. External leptotrichiae are absent. The septate desmosome at the level of the terminal cell is absent, but is present in the channel proximal cell at the level of terminal of cilia. The apical surface of the channel cell carries rare short microvilli. The basement membrane of cells of excretory channels forms deep invaginations almost reaching the apical membrane. Epithelium of excretory channels is deprived of cilia. Ultrastructure of cirtocytes and excretory channels of B. semperi is similar to those in representatives of the suborder Proseriata (Seriata). The significance of ultrastructure of the Proseriata cirtocytes, especially of the order of formation of versh, for construction of phylogeny of Platyhelminthes is discussed.

Seminal Fluid-Mediated Manipulation of Post-mating Behavior in a Simultaneous Hermaphrodite.

Seminal fluid proteins (SFPs) are uniquely positioned to mediate post-mating sexual selection and sexual conflict [1-3]. This role may be especially important in simultaneous hermaphrodites, in which individuals will often agree to receive sperm in order to be able to donate it, shifting the arena of sexual selection to post-mating reproductive interactions [4-7]. Nevertheless, as in separate-sexed organisms, identifying individual SFPs responsible for specific post-mating effects is difficult, owing to the complexity, rapid evolution, and functional redundancy of seminal fluid [8-11]. Here, we sought to identify SFPs that influence one striking post-mating behavior of the simultaneously hermaphroditic flatworm Macrostomum lignano, the so-called "suck behavior," in which worms respond to ejaculate receipt by placing their pharynx over their female genital opening and seemingly attempt to remove sperm and/or other ejaculate components [12-14]. We hypothesized that sucking is counter to the sperm donor's interests, potentially selecting for SFPs that reduce the suck propensity of mating partners. We tested this using a combination of quantitative genetics and RNA interference (RNAi) knockdown. As predicted, we found negative genetic correlations between the expression levels of six (out of 58) seminal fluid transcripts and partner suck propensity. RNAi knockdown confirmed that two of these transcripts, designated suckless-1 and suckless-2, indeed caused mating partners to suck less often. We suggest that these proteins are male counter-adaptations to recipient suck behavior, which itself is likely a female counter-adaptation in the ongoing evolutionary conflict to (re)gain control over ejaculate fate after mating in this hermaphroditic organism.

Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano.

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.

Characterization of the stem cell system of the acoel Isodiametra pulchra.

BACKGROUND: Tissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration. RESULTS: We established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation. CONCLUSIONS: We introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.