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Previous studies on sex allocation in simultaneous hermaphrodites have typically focused either on evolutionary or one-time, ontogenetic optimization of sex allocation, ignoring variation within an individual's lifetime. Here, we study whether hermaphrodites also possess facultative sex allocation, that is, a phenotypic flexibility, allowing them to distribute resources to either sex in an opportunistic way during their adult lifetime. We used the simultaneously hermaphroditic free-living flatworm Macrostomum lignano and raised individuals in pairs and groups of eight worms (further called octets) until sexual maturity was reached and sex allocation for the current conditions was expected to be set. Treatment groups were subsequently transferred to the alternative group size, that is, from pairs to octets or from octets to pairs, and compared to two control groups, which were transferred without changing group size. The results show that worms in treatment groups responded as expected by the local mate competition theory for simultaneous hermaphrodites: increasing group size resulted in a shift toward a more male-biased sex allocation and vice versa. These findings reveal that sex allocation in these animals is not fixed during ontogeny, but remains flexible after maturation. We argue that phenotypically flexible sex allocation in hermaphroditic animals may help us to understand the evolution and ecology of hermaphroditism.

The marine microturbellarian Macrostomum lignano (Platyhelminthes, Rhabditophora) is an emerging laboratory model used by a growing community of researchers because it is easy to cultivate, has a fully sequenced genome, and offers multiple molecular tools for its study. M. lignano has a compartmentalized brain that receives sensory information from receptors integrated in the epidermis. Receptors of the head, as well as accompanying glands and specialized epidermal cells, form a compound sensory structure called the frontal glandular complex. In this study, we used semi-serial transmission electron microscopy (TEM) to document the types, ultrastructure, and three-dimensional architecture of the cells of the frontal glandular complex. We distinguish a ventral compartment formed by clusters of type 1 (multiciliated) sensory receptors from a central domain where type 2 (collar) sensory receptors predominate. Six different types of glands (rhammite glands, mucoid glands, glands with aster-like and perimaculate granula, vacuolated glands, and buckle glands) are closely associated with type 1 sensory receptors. Endings of a seventh type of gland (rhabdite gland) define a dorsal domain of the frontal glandular complex. A pair of ciliary photoreceptors is closely associated with the base of the frontal glandular complex. Bundles of dendrites, connecting the receptor endings with their cell bodies which are located in the brain, form the (frontal) peripheral nerves. Nerve fibers show a varicose structure, with thick segments alternating with thin segments, and are devoid of a glial layer. This distinguishes platyhelminths from larger and/or more complex invertebrates whose nerves are embedded in prominent glial sheaths.

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.

The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227 and ∼220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84–85%), low BUSCO duplication rates (8.3–6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms.

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.

The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

The intestine is a multifunctional organ responsible for digestion, nutrient absorption, metabolic regulation, and innate immunity. In flatworms, recent studies have highlighted the importance of intestine-enriched genes expressed strongly in cells of the digestive tract. These genes are not only involved in digestion, nutrient uptake, transport, metabolism, and feeding behavior, but also in the modulating dynamics of stem cells (neoblasts). In , the molecular mechanisms regulating interaction between digestive and neural processes remain poorly understood, as in other free-living flatworms. Therefore, identifying the genes required for intestinal integrity and feeding behavior is essential for understanding the underpinning mechanisms. In this study, we examined intestine-enriched candidate genes predicted to be involved in cell differentiation and maintenance of the intestine in and whether the knockdown of these genes affects other tissues' functioning. Using RNAi-mediated gene silencing, we identified four genes ( , , , and ) whose knockdown causes pronounced phenotypes, including reduced feeding, fasting behavior, decreased body size and cell proliferation, low reproduction, and altered expression of an intestine-specific apob promoter. We have characterized their roles in intestinal homeostasis and neoblast dynamics and discussed potential mechanisms linking gene disruption to changes in feeding behavior.

Abstract Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

Expansion and losses of gene families are important drivers of molecular evolution. A recent survey of Fox genes in flatworms revealed that this superfamily of multifunctional transcription factors, present in all animals, underwent extensive losses and expansions during platyhelminth evolution. In this paper, I analyzed Fox gene complement in four additional species of platyhelminths, that represent early-branching lineages in the flatworm phylogeny: catenulids ( Stenostomum brevipharyngium and Stenostomum leucops ) and macrostomorphs ( Macrostomum hystrix and Macrostomum cliftonense ). Phylogenetic analysis of Fox genes from this expanded set of species provided evidence for multiple independent expansions of Fox gene families within flatworms. Notably, FoxG , a panbilaterian brain-patterning gene, appears to be the least susceptible to duplication, while FoxJ1 , a conserved ciliogenesis factor, has undergone extensive expansion in various flatworm lineages. Analysis of the single-cell atlas of S . brevipharyngium , combined with RNA in situ hybridization, elucidated the tissue-specific expression of the selected Fox genes: FoxG is expressed in the brain, three of the Fox genes ( FoxN2/3–2 , FoxO4 and FoxP1 ) are expressed in the pharyngeal cells of likely glandular function, while one of the FoxQD paralogs is specifically expressed in the protonephridium. Overall, the evolution of Fox genes in flatworms appears to be characterized by an early contraction of the gene complement, followed by lineage-specific expansions that have enabled the co-option of newly evolved paralogs into novel physiological and developmental functions.

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. Regeneration capable flatworms have emerged as powerful models for studying stem cell biology and patterning, however their study has been hindered by the lack of transgenesis methods. Here, the authors describe a transgenesis method for Macrostomum lignano , as well as a new annotated genome sequence.