Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano.

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

No evidence for strong cytonuclear conflict over sex allocation in a simultaneously hermaphroditic flatworm.

BACKGROUND: Cytoplasmic sex allocation distorters, which arise from cytonuclear conflict over the optimal investment into male versus female reproductive function, are some of the best-researched examples for genomic conflict. Among hermaphrodites, many such distorters have been found in plants, while, to our knowledge, none have been clearly documented in animals. METHODS: Here we provide a quantitative test for cytonuclear conflict over sex allocation in the simultaneously hermaphroditic flatworm Macrostomum lignano. We used a quantitative genetic breeding design, employing pair-wise crosses of 2 × 15 independent inbred lines, to partition the phenotypic variance in several traits (including sex allocation) into its nuclear and cytoplasmic components. RESULTS: Although the nuclear genetic background had a significant effect on all traits analyzed, we found significant cytoplasmic genetic variation only for ovary size, there explaining just 4.1% of the variance. A subsequent statistical power analysis showed that the experimental design had considerable power to detect cytonuclear interactions. CONCLUSION: We conclude that there were no strong effects of cytonuclear conflict in the studied populations, possibly because the usually compact mitochondrial genomes in animals have a lower evolvability than the large mitochondrial genomes in plants or because the sampled populations currently do not harbor variation at putative distorter and/or the restorer loci.

Indirect genetic effects and sexual conflicts: Partner genotype influences multiple morphological and behavioral reproductive traits in a flatworm.

The expression of an individual's phenotypic traits can be influenced by genes expressed in its social partners. Theoretical models predict that such indirect genetic effects (IGEs) on reproductive traits should play an important role in determining the evolutionary outcome of sexual conflict. However, empirical tests of (i) whether reproductive IGEs exist, (ii) how they vary among genotypes, and (iii) whether they are uniform for different types of reproductive traits are largely lacking. We addressed this in a series of experiments in the simultaneously hermaphroditic flatworm Macrostomum lignano. We found strong evidence for IGEs on both morphological and behavioral reproductive traits. Partner genotype had a significant impact on the testis size of focal individuals-varying up to 2.4-fold-suggesting that IGEs could mediate sexual conflicts that target the male sex function. We also found that time to first copulation was affected by a genotype × genotype interaction between mating partners, and that partner genotype affected the propensity to copulate and perform the postcopulatory suck behavior, which may mediate conflicts over the fate of received ejaculate components. These findings provide clear empirical evidence for IGEs on multiple behavioral and morphological reproductive traits, which suggests that the evolutionary dynamics of these traits could be altered by genes contained in the social environment.

Evidence for Karyotype Polymorphism in the Free-Living Flatworm, Macrostomum lignano, a Model Organism for Evolutionary and Developmental Biology.

Over the past decade, the free-living flatworm Macrostomum lignano has been successfully used in many areas of biology, including embryology, stem cells, sexual selection, bioadhesion and aging. The increased use of this powerful laboratory model, including the establishment of genomic resources and tools, makes it essential to have a detailed description of the chromosome organization of this species, previously suggested to have a karyotype with 2n = 8 and one pair of large and three pairs of small metacentric chromosomes. We performed cytogenetic analyses for chromosomes of one commonly used inbred line of M. lignano (called DV1) and uncovered unexpected chromosome number variation in the form of aneuploidies of the largest chromosomes. These results prompted us to perform karyotypic studies in individual specimens of this and other lines of M. lignano reared under laboratory conditions, as well as in freshly field-collected specimens from different natural populations. Our analyses revealed a high frequency of aneuploids and in some cases other numerical and structural chromosome abnormalities in laboratory-reared lines of M. lignano, and some cases of aneuploidy were also found in freshly field-collected specimens. Moreover, karyological analyses were performed in specimens of three further species: Macrostomum sp. 8 (a close relative of M. lignano), M. spirale and M. hystrix. Macrostomum sp. 8 showed a karyotype that was similar to that of M. lignano, with tetrasomy for its largest chromosome being the most common karyotype, while the other two species showed a simpler karyotype that is more typical of the genus Macrostomum. These findings suggest that M. lignano and Macrostomum sp. 8 can be used as new models for studying processes of partial genome duplication in genome evolution.

Quantifying episodes of sexual selection: Insights from a transparent worm with fluorescent sperm.

Sexual selection operates through consecutive episodes of selection that ultimately contribute to the observed variance in reproductive success between individuals. Understanding the relative importance of these episodes is challenging, particularly because the relevant postcopulatory fitness components are often difficult to assess. Here, we investigate different episodes of sexual selection on the male sex function, by assessing how (precopulatory) mating success, and (postcopulatory) sperm-transfer efficiency and sperm-fertilizing efficiency contribute to male reproductive success. Specifically, we used a transgenic line of the transparent flatworm, Macrostomum lignano, which expresses green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking and paternity analysis. We found that a large proportion of variance in male reproductive success arose from the postcopulatory episodes. Moreover, we also quantified selection differentials on 10 morphological traits. Testis size and seminal vesicle size showed significant positive selection differentials, which were mainly due to selection on sperm-transfer efficiency. Overall, our results demonstrate that male reproductive success in M. lignano is not primarily limited by the number of matings achieved, but rather by the ability to convert matings into successful fertilizations, which is facilitated by producing many sperm.

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

The first multi-gene phylogeny of the Macrostomorpha sheds light on the evolution of sexual and asexual reproduction in basal Platyhelminthes.

The Macrostomorpha-an early branching and species-rich clade of free-living flatworms-is attracting interest because it contains Macrostomum lignano, a versatile model organism increasingly used in evolutionary, developmental, and molecular biology. We elucidate the macrostomorphan molecular phylogeny inferred from both nuclear (18S and 28S rDNA) and mitochondrial (16S rDNA and COI) marker genes from 40 representatives. Although our phylogeny does not recover the Macrostomorpha as a statistically supported monophyletic grouping, it (i) confirms many taxa previously proposed based on morphological evidence, (ii) permits the first placement of many families and genera, and (iii) reveals a number of unexpected placements. Specifically, Myozona and Bradynectes are outside the three classic families (Macrostomidae, Microstomidae and Dolichomacrostomidae) and the asexually fissioning Myomacrostomum belongs to a new subfamily, the Myozonariinae nov. subfam. (Dolichomacrostomidae), rather than diverging early. While this represents the first evidence for asexuality among the Dolichomacrostomidae, we show that fissioning also occurs in another Myozonariinae, Myozonaria fissipara nov. sp. Together with the placement of the (also fissioning) Microstomidae, namely as the sister taxon of Dolichomacrostomidae, this suggests that fissioning is not basal within the Macrostomorpha, but rather restricted to the new taxon Dolichomicrostomida (Dolichomacrostomidae+Microstomidae). Furthermore, our phylogeny allows new insights into the evolution of the reproductive system, as ancestral state reconstructions reveal convergent evolution of gonads, and male and female genitalia. Finally, the convergent evolution of sperm storage organs in the female genitalia appears to be linked to the widespread occurrence of hypodermic insemination among the Macrostomorpha.

Fluorescent sperm in a transparent worm: validation of a GFP marker to study sexual selection.

BACKGROUND: Sexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement. RESULTS: GFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel's laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm donors can displace previously stored sperm and replace it with their own. CONCLUSION: The availability of the GFP-techniques in a transparent organism provide unique opportunities to visualise and quantify internal processes in the female reproductive tract after mating, which opens new avenues in the study of sexual selection.

Sex allocation adjustment to mating group size in a simultaneous hermaphrodite.

Sex allocation theory is considered as a touchstone of evolutionary biology, providing some of the best supported examples for Darwinian adaptation. In particular, Hamilton's local mate competition theory has been shown to generate precise predictions for extraordinary sex ratios observed in many separate-sexed organisms. In analogy to local mate competition, Charnov's mating group size model predicts how sex allocation in simultaneous hermaphrodites is affected by the mating group size (i.e., the number of mating partners plus one). Until now, studies have not directly explored the relationship between mating group size and sex allocation, which we here achieve in the simultaneously hermaphroditic flatworm Macrostomum lignano. Using transgenic focal worms with ubiquitous expression of green-fluorescent protein (GFP), we assessed the number of wild-type mating partners carrying GFP+ sperm from these focal worms when raised in different social group sizes. This allowed us to test directly how mating group size was related to the sex allocation of focal worms. We find that the proportion of male investment initially increases with increasing mating group size, but then saturates as predicted by theory. To our knowledge, this is the first direct test of the mating group size model in a simultaneously hermaphroditic animal.

Phenotypic engineering of sperm-production rate confirms evolutionary predictions of sperm competition theory.

Sperm production is a key male reproductive trait and an important parameter in sperm competition theory. Under sperm competition, paternity success is predicted to depend strongly on male allocation to sperm production. Furthermore, because sperm production is inherently costly, individuals should economize in sperm expenditure, and conditional adjustment of the copulation frequency according to their sperm availability may be expected. However, experimental studies showing effects of sperm production on mating behaviour and paternity success have so far been scarce, mainly because sperm production is difficult to manipulate directly in animals. Here, we used phenotypic engineering to manipulate sperm-production rate, by employing dose-dependent RNA interference (RNAi) of a spermatogenesis-specific gene, macbol1, in the free-living flatworm Macrostomum lignano. We demonstrate (i) that our novel dose-dependent RNAi approach allows us to induce high variability in sperm-production rate; (ii) that a reduced sperm-production rate is associated with a decreased copulation frequency, suggesting conditional adjustment of mating behaviour; and (iii) that both sperm production and copulation frequency are important determinants of paternity success in a competitive situation, as predicted by sperm competition theory. Our study clearly documents the potential of phenotypic engineering via dose-dependent RNAi to test quantitative predictions of evolutionary theory.

Mating behavior and the evolution of sperm design.

Sperm are the most diverse of all animal cell types, and much of the diversity in sperm design is thought to reflect adaptations to the highly variable conditions under which sperm function and compete to achieve fertilization. Recent work has shown that these conditions often evolve rapidly as a consequence of multiple mating, suggesting a role for sexual selection and sexual conflict in the evolution of sperm design. However, very little of the striking diversity in sperm design is understood functionally, particularly in internally fertilizing organisms. We use phylogenetic comparative analyses covering 16 species of the hermaphroditic flatworm genus Macrostomum to show that a complex sperm design is associated with reciprocal mating and that this complexity is lost secondarily when hypodermic insemination--sperm injection through the epidermis--evolves. Specifically, the complex sperm design, which includes stiff lateral bristles, is likely a male persistence trait associated with sexual conflicts over the fate of received ejaculates and linked to female resistance traits, namely an intriguing postcopulatory sucking behavior and a thickened epithelium of the sperm-receiving organ. Our results suggest that the interactions between sperm donor, sperm, and sperm recipient can change drastically when hypodermic insemination evolves, involving convergent evolution of a needle-like copulatory organ, a simpler sperm design, and a simpler female genital morphology. Our study documents that a shift in the mating behavior may alter fundamentally the conditions under which sperm compete and thereby lead to a drastic change in sperm design.

Male-biased sex-ratio distortion caused by Octosporea bayeri, a vertically and horizontally-transmitted parasite of Daphnia magna.

Female-biased sex-ratio distortion is often observed in hosts infected with vertically-transmitted microsporidian parasites. This bias is assumed to benefit the spread of the parasite, because male offspring usually do not transmit the parasite further. The present study reports on sex-ratio distortion in a host-parasite system with both horizontal and vertical parasite transmission: the microsporidium Octosporea bayeri and its host, the planktonic cladoceran Daphnia magna. In laboratory and field experiments, we found an overall higher proportion of male offspring in infected than in uninfected hosts. In young males, there was no parasite effect on sperm production, but, later in life, infected males produced significantly less sperm than uninfected controls. This shows that infected males are fertile. As males are unlikely to transmit the parasite vertically, an increase in male production could be advantageous to the host during phases of sexual reproduction, because infected mothers may obtain uninfected grandchildren through their sons. Life-table experiments showed that, overall, sons harboured more parasite spores than their sisters, although they reached a smaller body size and died earlier. Male production may thus be beneficial for the parasite when horizontal transmission has a large pay-off as males may contribute more effectively to parasite spread than females.

Mixed inoculations of a microsporidian parasite with horizontal and vertical infections.

Mixed infections, where more than one parasite genotype is present in a single host, have been suggested to be an important factor in host-parasite interactions. As the host represents a limited resource, co-infecting parasite genotypes are expected to be under resource competition. Competition will not only modify the survival of the co-infecting genotypes, but is also likely to affect total within-host parasite growth as well as host survival and reproduction. We measured parasite infectivity and spore production of seven isolates of Octosporea bayeri (Microsporidia) and their effect on the reproduction and longevity of its host Daphnia magna (Cladocera), after single- or double-isolate inoculations through vertical and horizontal transmission. Double-isolate inoculations increased parasite infectivity and total spore production in horizontal infections, but had no significant effect on host reproduction or longevity. The increase in spore production in double-isolate inoculations was not found when infections occurred vertically. Our results suggest that, depending on the way the infection was acquired, within-host reproduction can increase as a result of parasite genetic diversity, without necessarily affecting the host's fitness. Whether this challenges the current views of virulence evolution depends on the definitions used.

Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna.

Estimates of phenoloxidase (PO) activity have been suggested as a useful indicator of immunocompetence in arthropods, with the idea that high PO activity would indicate high immunocompetence against parasites and pathogens. Here, we test for variation in PO activity among clones of the planktonic crustacean Daphnia magna and its covariation with susceptibility to infections from four different microparasite species (one bacterium and three microsporidia). Strong clonal variation in PO activity was found within and among populations of D. magna, with 45.6% of the total variation being explained by the clone effect. Quantitative measures of parasite success in infection correlated negatively with PO activity when tested across four host populations. However, these correlations disappeared when the data were corrected for population effects. We conclude that PO activity is not a useful measure of resistance to parasites or of immunocompetence within populations of D. magna. We further tested whether D. magna females that are wounded to induce PO activity are more resistant to infections with the bacterium Pasteuria ramosa than non-wounded controls. We found neither a difference in susceptibility nor a difference in disease progression between the induced group and the control group. These results do not question the function of the PO system in arthropod immune response, but rather suggest that immunocompetence cannot be assessed by considering PO activity alone. Immune response is likely to be a multifactorial trait with various host and parasite characteristics playing important roles in its expression.