Sex allocation theory for simultaneous hermaphrodites predicts an influence of the mating group size on sex allocation. Mating group size may depend on the size of the group in which an individual lives, or on the density, but studies to date have not distinguished between the two factors. We performed an experiment in which we raised a transparent simultaneous hermaphrodite, the flatworm Macrostomum sp., in different group sizes (pairs, triplets, quartets and octets) and in different enclosure sizes (small and large). This design allows us to differentiate between the effects of group size and density. After worms reached maturity we determined their reproductive allocation patterns from microscopic images taken in vivo. The results suggest that the mating group size is a function of the group size, and not of the density. They support the shift to higher male allocation in larger mating groups predicted by sex allocation theory. To our knowledge, this is the first study that unambiguously shows phenotypically plastic sex allocation in response to mating group size in a simultaneous hermaphrodite.

Stem cells (neoblasts) in Platyhelminthes are pluripotent, and likely totipotent, undifferentiated cells which retain throughout adult life the capacity to proliferate and from which all somatic cells as well as the germ cells derive. However, basic data on the pool and heterogeneity of neoblasts, their rates of differentiation into sets and subsets of differentiated cells, and their migration to different body regions are still lacking. To fill this gap, S-phase cells in the macrostomid Macrostomum sp. were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU). S-phase cells were found to be neoblasts and to be distributed in two bands along the lateral sides of the body leaving unlabeled the median axis of the body and the region anterior to the eyes. This distribution is parallel to that of mitotic cells demonstrated using an antibody to phosphorylated histone H3. At different chase times, clusters of BrdU-labeled cells appear, labeled cells migrate to formerly unlabeled areas, and they differentiate into several somatic cell types and into germ cells. Finally, continuous exposure to BrdU shows an extensive renewal of the epithelial cells. Altogether, these results strengthen the idea of platyhelminth neoblasts as an unparalleled stem-cell system within the Animal Kingdom calling for further investigation.

A new model organism among the lower Bilateria and the use of digital microscopy in taxonomy of meiobenthic Platyhelminthes: Macrostomum lignano, n. sp. (Rhabditophora, Macrostomorpha) Abstract Macrostomum lignano n. sp. is a member of the Macrostomorpha, the basal-most subtaxon of the Platyhelminthes-Rhabditophora. This new species can be easily cultured in the laboratory and has been already the subject of several developmental/evolutionary studies. The small size, with only about 25 000 cells constituting the major bilaterian organ systems, makes this simultaneous hermaphrodite a possible candidate for a new model organism that is phylogenetically more basal than any of the model organisms currently used in such studies within the Bilateria. M. lignano belongs to the largest genus of the Macrostomorpha. Over 100 marine, fresh water and brackish water species are contained in the genus Macrostomum, some of them with worldwide distribution pattern. Within it, M. lignano is a member of the M. tuba-species group, which we have summarized here. In the species description, we have used a novel approach to document such small soft-bodied meiobenthic organisms: we provide extensive digital micrographical documentation, which are deposited as a CD together with the type material.

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.

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2 0 -O-methyl modification at their 3 0 end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3 0 -5 0 exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.