BMC Biology

Choanoflagellate genetics has undergone rapid and impactful developments in the last decade. Currently, the primary method for genetic modification of choanoflagellates relies on proprietary nucleofection reagents to deliver transgenes for ectopic expression or CRISPR-Cas9 ribonucleoprotein complexes for targeted genome editing. The acquisition of proprietary buffers required for nucleofection can hamper advances in choanoflagellate research due to costs, shipping limitations, and restrictions that prevent buffer components from being optimized for understudied organisms. Therefore, we test whether a low-cost in-house electroporation buffer developed for other systems can replace the proprietary buffer currently used for choanoflagellate transfection. Here, we present an in-house buffer with transfection efficiency comparable to that of the previously established proprietary buffer. This work increases the accessibility of choanoflagellate genetics and can broaden research participation in investigating animal origins.

BackgroundMyxozoans are ancient cnidarian parasites with highly derived genomes characterized by an extremely accelerated rate of nucleotide substitution, abundant orphan and lineage-specific genes without further characterization. Genome data are limited to two out of the four main evolutionary lineages, assemblies are highly fragmented and often show significant levels of host contamination. ResultsWe present a near chromosome-scale myxozoan genome, based on Oxford Nanopore-reads of Sphaerospora molnari, a member of the previously uncharacterized group of blood-feeding myxozoans, thereby addressing a key gap in the subphylums phylogeny. The haploid genome assembly spans 40.17 Mb in 40 contigs, harbors 14,957 genes and shows the smallest mitogenome of myxozoans (14,015 bp). Gene gain/gene loss analyses showed that myxozoans have small ancestral gene repertoires and show highly lineage-specific genome compositions. Using comparative analyses focusing on identifying unique but diversified gene compartments in S. molnari, we discovered taxonomically restricted protist genes related to red blood cell attachment (Plasmodium ETRAMPs) and surface protein variation (Plasmodium variant surface antigen families RIFIN and STEVOR, as well as Metamonada variant-specific surface proteins, VSPs), raising questions about their origins and evolution. A genomic trait shared between several myxozoans is the significant expansion of DNA transposable elements belonging to the mutator-like elements (MULEs), and while the simple copy-paste mechanism of these transposases may suggest frequent uncontrolled mutation, we demonstrate domestication of MULEs into transcription factors. Analyses of the gene fragments of chimeric MULEs (pack-MULES) of S. molnari show that these coincide with highly diversified gene groups in this lineage, including alien genes, suggesting MULEs as a driving force for gene evolution in myxozoans. ConclusionsParasitic lifestyle shifts drive exceptionally rapid genome evolution in myxozoans, primarily through nonadaptive mutation and gene transfer via MULEs, with adaptive refinement through MULE domestication and selection. In S. molnari, these processes underpin unique erythrocyte exploitation and immune evasion strategies essential for survival in the hosts bloodstream.

All cells of a multicellular organism usually share an identical genome, faithfully transmitted through successive divisions. Yet, a number of animal species deviate from this dogma, as parts of their DNA are systematically eliminated in all their somatic nuclei, in a process called Programmed DNA Elimination (PDE). PDE leads to the unexpected reorganisation of the genome at every generation in all somatic cells but its molecular mechanism, evolutionary origins, and functional significance remain unknown. This lack of understanding partially stems from limitations in genetically tractable model species. PDE can target an entire chromosome, or involve chromosome fragmentation followed by selective fragment retention and elimination, raising further questions on genome stability, genome integrity and mechanisms of DNA repair. PDE by chromosome fragmentation has been described in parasitic nematodes in the family Ascarididae, copepods in the genus Cyclops and unicellular ciliates. More recently, PDE has been discovered in three non-parasitic, lab-tractable nematode species from the Rhabditidae family, opening new perspectives. In this study, we used cytological approaches to screen 25 new Rhabditidae species for PDE. We found evidence of PDE in 17 species. Our work reveals that PDE is present in 12 out of 17 tested genera, demonstrating its widespread presence in Rhabditidae nematodes, with the notable exception of C. elegans. Genetic tools have already been established for some species. This work provides a collection of lab-tractable species that can be used to test many aspects of somatic Programmed DNA Elimination by chromosome fragmentation in animals.

Nociception is an essential response for organisms to avoid potential harm and promote survival. Its molecular determinants are largely conserved across Eumetazoa. TRPV receptors are polymodal ion channels exhibiting selective peripheral expression and functional coupling that underpins nociception and pain modulation in complex organisms. However, the execution of protective behaviours triggered by TRPVs is also found in species with a simpler nervous organisation, thus encouraging their investigation in invertebrate model organisms to increase understanding of animal nociception. Cephalopods represent an interesting invertebrate phylum with respect to the evolution of the nervous system, whose complexity suggests it might support pain-like states that exist in vertebrates. This possibility is reflected by the inclusion of cephalopods in the UK and EU animal welfare legislations. Despite this, there is poor characterisation of cephalopod molecular nociceptors. For this reason, we used in silico analysis to identify two TRPV channels in Octopus vulgaris genome (Ovtrpv1 and Ovtrpv2). We validated the putative transcript sequences and highlighted prevalent expression in sensory tissues. We investigated the functional competence of these TRPVs by heterologously expressing Ovtrpv1 and Ovtrpv2 cDNA into Caenorhabditis elegans null mutants of the orthologous genes, ocr-2 and osm-9 respectively. Ovtrpvs successfully rescued the aversive response to chemical and mechanical noxious stimuli in the C. elegans mutants, suggesting these receptors are polymodal nociceptors. Additionally, complementary investigation using Xenopus laevis oocytes showed Ovtrpv1 and Ovtrpv2 form an active heteromeric channel gated by nicotinamide. This study highlights Ovtrpvs as an important route to better understand nociceptive detection in cephalopods.

Infertility generates profound psychological and social distress for both women and men, yet mens communicative experiences remain comparatively underexamined. Male infertility (MI) is often shaped by stigma, norms of masculinity, and limited opportunities for emotional disclosure, constraining help-seeking in offline settings. This study investigates how men use anonymous online peer-support spaces to discuss MI by analyzing discussions from the r/maleinfertility subreddit on Reddit. Using natural language processing techniques, we examined 10,769 posts and 80,381 comments published between 2013 and 2025. Analyses assessed sentiment and emotional expression, topic structure, hyperlink networks, and discussions related to diagnostic testing, treatment decision-making, and donor sperm use. Topic modeling revealed a functional differentiation between posts and comments. Original posts primarily focused on clinical sense-making, including interpretation of semen analyses, hormonal testing, and assisted reproduction options. In contrast, comments emphasized emotional validation, experiential knowledge-sharing, and normalization of alternative family-building pathways. Emotional expression varied by discussion topic, with heightened fear and sadness in conversations involving genetic testing, surgical sperm retrieval, and donor sperm. Hyperlink analysis indicated frequent engagement with peer-reviewed medical information, reflecting active evidence-seeking alongside peer exchange. Taken together, findings suggest that anonymous online communities function as critical infrastructures of support for men experiencing infertility, enabling forms of disclosure and vulnerability often constrained in offline contexts. These spaces facilitate interpretation of medical information, collective coping, and decision-making regarding treatment and donor options. The study highlights the role of digital anonymity in mitigating stigma and expanding communicative possibilities for men navigating infertility alongside clinical care.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, impacting hundreds of millions of people and animals globally. Disease pathology primarily originates from host immune responses to parasite eggs, which are produced only when female schistosomes are continuously paired with males. Past research focused on pairing-dependent female sexual maturation, while scarce data exist for the males reproductive biology. In this study, we characterized the G protein-coupled receptor Smgpcr9 (Smp_244240), an orphan Class A (Rhodopsin-like) GPCR with a testis-preferential and pairing-influenced expression profile in S. mansoni males. Previous bulk RNA-seq analyses of adult worms and their isolated gonads revealed that Smgpcr9 belongs to a subgroup of GPCR genes with abundant testis-preferential and pairing-influenced transcript levels in males but low and extremely low expression in unpaired and paired females, respectively. This male-/unpaired female-biased expression pattern mirrors that of neuropeptide (npp) genes of S. mansoni such as Smnpp26 and Smnpp41. In a deorphanization approach using yeast-two-hybrid analyses, GPCR internalization experiments, bioluminescence resonance energy transfer assays, and by modeling and docking analyses, we provide first evidence that both NPPs can interact with SmGPCR9. Furthermore, we optimized a GPCR RNAi approach and achieved efficient transcript knockdown (> 90%) enabling robust functional characterization of Smgpcr9. Following RNAi, physiological and morphological analyses revealed that SmGPCR9 regulates key aspects of male reproductive biology like testis morphology and spermatogenesis. Remarkably, ovary structure and egg production were also affected in paired females post RNAi. We observed similar phenotypes plus motility constraints and reduced stem-cell proliferation in both sexes upon RNAi of Smnpp26 and Smnpp41. In all cases, RNAi downstream analyses by RT-qPCR of marker genes substantiated the observed phenotypic effects. These results strongly indicate the importance of SmGPCR9, SmNPP26, and SmNPP41 for spermatogenesis and further physiological processes in male and female S. mansoni. Author SummaryResearch of the reproductive biology of schistosomes focused mainly on females so far, which upon pairing sexually mature to produce eggs that are important for the life cycle maintenance but also for the pathogenesis of schistosomiasis, the infectious disease caused by these parasites. We investigated a yet unknown G protein-coupled receptor, Smgpcr9, which showed a testis-preferential and pairing-influenced expression profile in Schistosoma mansoni males. To this end, we optimized an RNA interference (RNAi) approach for knockdown analysis, identified neuropeptides (NPPs) as potential ligands by different biochemical approaches and modeling and docking analyses, and we investigated the roles of SmGPCR9 and two interacting NPPs, SmNPP26 and SmNPP41, by physiological, microscopical, and molecular techniques. Our results strongly suggest that SmGPCR9 and both NPPs regulate spermatogenesis. Furthermore, we detected effects on ovary morphology, egg production, and stem-cell proliferation of paired females post RNAi. Taken together, we deorphanized SmGPCR9 and showed for the first time the essential role of a so far uncharacterized GPCR and two interacting neuropeptides for spermatogenesis. Our results shed first light on spermatogenesis regulatory processes controlled by GPCRs and neuropeptides in male S. mansoni and thus expand our understanding of the roles of GPCR-NPP signaling for schistosome reproductive biology.

In asexual reproduction, meiosis must be bypassed or altered to maintain ploidy from mother to daughter without fertilization. Most of the ways meiosis can be modified to this end are expected to reduce heterozygosity within individuals; however, many asexual species are highly heterozygous. Asexual reproduction is especially common among species of microscopic, desiccation-tolerant animals such as rotifers, nematodes, and tardigrades, but the cellular and genetic mechanisms underlying asexual reproduction have not been definitively documented in any species of tardigrade. Here, we show that the asexual tardigrade Hypsibius exemplaris fails to complete the cell division of meiosis I, followed by a complete meiosis II-like division, and reproduction proceeds without detectable loss of heterozygosity. We used a combined cytological and genomic approach to characterize the mechanism of reproduction and pattern of allele inheritance in this species. Furthermore, we identified heterozygous variants in a subset of transcriptionally active genes consistent with loss of function in one allele, suggesting that maintained heterozygosity in this species allowed divergence between alleles over time. This work establishes the meiotic mechanism and inheritance pattern of reproduction in H. exemplaris, which provides a framework for interpreting genetic variation in this organism as a laboratory model. Additionally, our finding that meiosis is modified in H. exemplaris via a mechanism that maintains heterozygosity across the genome adds to a growing body of evidence that maintaining heterozygosity is not detrimental to the long-term survival of asexual eukaryotes. Article SummaryAnimals that reproduce asexually must alter meiosis, a highly conserved process of two cell divisions normally used to make eggs and sperm. This study represents the first combined cytological and genetic characterization of how meiosis is modified in a tardigrade. The authors found that the model tardigrade Hypsibius exemplaris modifies meiosis by skipping the first cell division, but completing the second. Additionally, they found that this species preserves heterozygosity across the genome and from generation to generation. Finally, some genes show evidence of sequence divergence between alleles, supporting a broader conclusion that maintaining heterozygosity influences how asexual species genomes evolve.

Eukaryotic genomic DNA is packaged in the nucleus as chromatin - a DNA-protein aggregate regulating genome function, including transcription. Chromatin is classified as either active euchromatin or silent heterochromatin, with each marked by distinct histone post-translational modifications (PTMs). Chromatin composition also mediates genome organization, including how heterochromatin aggregates at the nuclear periphery while euchromatin localizes to the nucleus center. In fungi, heterochromatic loci cluster, including independent centromere and telomere clusters that form the Rabl chromosome conformation. However, it is unknown if chromatin composition and genome organization are conserved in closely related fungi, and how they are impacted by large-scale chromosomal rearrangements. Here, we examined differences in histone PTM deposition, gene expression, and genome organization in two yeast species from the order Pichiales, which diverged from the common ancestor shared with Saccharomyces cerevisiae more than 200 million years ago. We focused on Ogataea polymorpha, which is used for industrial protein production, and Ogataea haglerorum, an isolate of which harbors a translocation between chromosomes 1 and 6. We show that the enrichment of three activating PTMs - the trimethylation of lysine 4 of histone H3 (H3K4me3) and the acetylation of lysine 9 of histone H3 (H3K9ac) or lysine 16 of histone H4 (H4K16ac) - are similar genome-wide yet individual gene orthologs have distinct chromatin and expression patterns. While both Ogataea genomes organize into a Rabl conformation, the O. haglerorum translocation alters subtelomeric chromatin composition and expression of genes affected by the translocation. Our work highlights the genome function differences that occur on a microevolutionary scale.

BACKGROUNDDuring host colonization, fungal plant pathogens secrete effector-like proteins that alter host cell physiology and target plant-associated microbes. However, rapid evolution and low sequence conservation hinder the study and characterization of these proteins. The fungus Zymoseptoria passerinii infects Hordeum spp. and includes lineages adapted to wild and domesticated barley. To date, the evolution of effector-like proteins in this species has not been addressed. RESULTSWe combined a set of protein structure-based and network analyses to unravel the secretome of Z. passerinii. We first compared AlphaFold2 and ESMFold predictions to establish the baseline for structural analyses. We identified 72 structural clusters in the secretome, revealing fold-level relationships across divergent sequences. We showed that effector-like proteins with host putative immune-interfering functions evolved from a limited group of protein folds, whereas proteins with predicted antimicrobial properties were distributed across fold groups. Physicochemical comparisons indicate that antimicrobial effectors predominantly emerged through amino acid replacements on common effector-enriched scaffolds in Z. passerinii, reconfiguring surface charge and electrostatics. We analyzed intra- and interspecific structural variation in selected effector-enriched families by comparing Z. passerinii proteins and homologs in the sister species Z. tritici. We describe high structural similarity in core folds, with local variation in loop and surface-exposed regions, consistent with fold stability still enabling functional diversification. CONCLUSIONSThe secretome of Z. passerinii is organized around common structural folds that support diverse biological roles, including host manipulation and host-associated microbial interactions. Conserved scaffolds combined with local physicochemical variation likely contribute to rapid adaptive evolution in Z. passerinii.

Protein subcellular localisation is informative for understanding potential protein function, particularly in highly structured unicellular eukaryotes. Microscopy is especially powerful for interrogating localisation, providing high resolution single cell data about where a protein resides. We previously generated the TrypTag dataset - a genome-wide protein localisation resource for the human unicellular parasite Trypanosoma brucei using fluorescent protein tagging. This is a puissant dataset due to its scale: Originally captured with high content image analysis in mind, it is a formidable resource for machine learning or artificial intelligence tool development and testing. Here, we describe a Python module for programmatic access to this data rich resource. Images of each tagged cell line, together with segmented cell masks, can be accessed arbitrarily by gene ID and tagging terminus, the database can be searched by protein localisation, and tools are provided to assist foundational image analysis of individual T. brucei cell cycle stage and morphology. We stress-tested this tool by using it to examine a key feature of T. brucei morphogenesis during division: The old and newly formed flagellum and associated organelles tend to have different protein compositions, and using the TrypTag toolkit we show that there is extensive age-based differential content of these organelles while the daughter nuclei completely lack such asymmetry.

Neuropeptide signalling is transversally important in all living animals as it constitutes the basis of cellular communication. The investigation of the functional roles of peptide signalling represents an important route to understanding evolution of specific physiological traits and behaviours in metazoans. Allatostatins and their cognate receptors are classically defined as invertebrate neuropeptide hormones. Among these, allatostatin C was firstly associated with insect development. However, accumulating evidence recognises the presence of allatostatin C as a conserved signalling molecule across all invertebrate lineages, with reported functions spanning from regulation of feeding and digestion to immune responses and modulation of core nociception. Here we combined in silico and experimental approaches to reveal the interacting molecular determinants of the allatostatin C signalling in the cephalopod Octopus vulgaris, a scientifically and culturally interesting invertebrate for its centralised nervous system, capable of top-down modulation of complex behaviours. This resolved a single prepropeptide encompassing allatostatin C peptide (OvAstC), whose conserved mature form (AVITACYFQAVSCY) was shown to differentially activate two identified cognate receptors (OvAstCR1 and OvAstCR2) when heterologously expressed in the recombinant system HEK293G5A. PCR analysis carried out in O. vulgaris tissues, showed a broad distribution of OvAstC and OvAstCRs. This wide expression across nervous, immune and digestive tissues is consistent with a pleiotropic role of this peptidergic system. Together, the opioid/somatostatin-related phylogenetic placement of OvAstCRs and the broad expression of OvAstC components in nervous and sensory tissues nominate this pathway as a candidate for neuromodulatory control of sensory processing, including nociception, with potential welfare relevance in cephalopods. Significance statementCephalopods represent an evolutionarily distinctive molluscan lineage that evolved a centralised nervous system capable of displaying advanced learning and behavioural complexity compared with other invertebrates. These features, speculated to allow elaboration of pain-like states, granted cephalopods inclusion as the only invertebrate taxon requiring protection under European legislations when used in research. Investigation of the neuropeptide signalling in cephalopods is currently understudied despite its crucial role in regulating broad physiological functions in organisms. This study identified for the first time a single allatostatin C peptide and two cognate receptors in Octopus vulgaris. Our characterisation of a putative endogenous allatostatin C system in octopus, the accumulating evidence of its central role in invertebrate antinociception and its evolutionary relationship with the vertebrate-exclusive analgesic opioid family, represent a critical starting point for a more in-depth analysis of the physiological role of allatostatin C in this subclass of molluscs, with important welfare implications.

We often think of fungi as mysterious organisms that do not follow the general principles of other eukaryotes. Thus, when exciting results are found, these organisms do not always receive the rigorous level of scrutiny seen in other fields. For many fungal species, dispersal and reproduction relies on spores, either sexual or asexual. These spores can either have a single nucleus, or multiple nuclei, and the purpose of these presumably mitotic copies was unclear. Recently it was described that the multiple nuclei in these spores are not mitotic duplicates, but instead they share a single haploid set of chromosomes distributed across nuclei. Here, we provide fluorescent microscopy and UV mutagenesis data that is inconsistent with this hypothesis. Contrasting these previous results, we observe multiple sets of chromosomes in spores of both B. cinerea and N. crassa. We also observed a strong linear relationship between the number of nuclei in spores and the total acriflavine fluorescence, further supporting mitotic copies. Genome sequencing of colonies arising from UV-irradiated colonies also recovered variants at intermediate frequences, instead of the fixed 100% expected from the new model proposed. This evidence suggests that, as long suspected, these nuclei are indeed mitotic copies, and that a re-evaluation of fungal biology is not currently necessary.

BackgroundIn recent years, the rise of AI-mediated technological assistance has impacted applied mycology. Various tools employ AI to analyze images of Macromycetes fruiting bodies for species identification. This trend has sparked widespread interest in online applications despite the potential risks associated with relying on AI-generated advice. MethodsWe conducted a comparative analysis of popular AI-based mushroom identification tools using over 100 original photographs of fungi fruiting bodies from nearly 60 species, taken in real-world conditions. Reference searches were conducted with mushroom names in five languages, including Latin species names. Functional test scores and an overall accuracy score were calculated for twelve selected AI applications to evaluate their general reliability. ResultsThe AI-based applications evaluated in the study were able to recognize only a portion of the provided mushroom images. Even the best-performing tools frequently failed to accurately identify fruiting bodies in real-world conditions. None of the tested applications consistently provided a single, correct species name. Instead, users were often presented with multiple options, among which the correct answer might have been found. ConclusionWhen addressing mycological queries, it is crucial to recognize the inherent risk of relying solely on AI-mediated resources for mushroom identification. Various limitations hinder their effectiveness in real-world environments. These tools should only be viewed as supplementary aids since they are inadequate for making definitive or safety-critical decisions.

Menstrual cycles are major biological events with extensive effects on the brain and cognition, experienced by half of the human population. To develop a comprehensive account of human cognition, it is necessary to successfully integrate and characterize menstrual cycle effects in cognitive science research. However, current approaches to menstrual cycle analysis suffer from low data resolution and are not well-equipped to capture the highly variable, non-linear changes in outcomes of interest across the cycle. We present a validated standardized method remedying these issues, demonstrate its utility using hormonal, behavioral, and neuroimaging data, and provide an open-source toolkit to facilitate its use.

Traumatic brain injury (TBI) is a public health burden with short-term and long-term consequences for those who survive. Repetitive TBI (rTBI) is highly associated with neurodegenerative diseases such as chronic traumatic encephalopathy. Here, we present a force-adjustable, enhanced CO2-driven Drosophila model of rTBI to investigate locomotor, cognitive, proteomic, and neurodegenerative changes following rTBI. Injured males demonstrate dose-dependent locomotor deficits, while injured females display greater tolerance to rTBI. However, both sexes exhibit significant decision-making deficits. Proteomic analysis revealed changes in proteins linked with locomotory behavior, mitochondria, and energy production post-injury, correlating with observed behavioral phenotypes. Histological analysis revealed increased vacuolization area in rTBI flies. These results suggest that rTBI drives sex-specific behavioral deficits, proteomic disruption, and neurodegeneration. Overall, this enhanced Drosophila rTBI model provides a valuable tool for exploring sexually dimorphic outcomes and understanding the long-term pathophysiology of repetitive brain trauma in the context of neurodegenerative disease. Significance StatementRepetitive traumatic brain injury (rTBI) is linked to long-term cognitive decline and neurodegeneration, yet standardized, scalable models that capture sex-dependent outcomes are limited. We developed an automated, force-adjustable, closed-head Drosophila rTBI apparatus that delivers reproducible injuries without off-target damage, enabling precise control of injury severity across sexes. Using this platform, we show sexually dimorphic survival and locomotor vulnerability, persistent decision-making deficits, and progressive brain pathology marked by vacuolization and apoptosis. Proteomic profiling reveals time-dependent, sex-specific disruption of mitochondrial, oxidative stress, and locomotor-related protein networks, providing mechanistic entry points for discovering modifiers and therapies for repetitive brain trauma.

This protocol generates gregarious and solitarious density-dependent phenotypes in multiple Schistocerca species under controlled environmental conditions. It describes cage setup, feeding, animal handling, and sterile dissection workflows to isolate nervous, chemosensory, gut, fat body, and female reproductive tissues from nymphs and adults. It emphasizes rapid tissue stabilization and RNase-control practices for downstream single-tissue DNA and RNA analyses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/705994v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@119ec2forg.highwire.dtl.DTLVardef@e115b7org.highwire.dtl.DTLVardef@158ad1dorg.highwire.dtl.DTLVardef@cd54d7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Orphan genes, lacking homologs in other species, are systematically found across genomes. Their presence may result from extensive divergence from pre-existing genes or from de novo gene birth, which occurs when a gene emerges from a previously non-genic region. In this study, we identified orphan genes in the genomes of globally distributed plant-parasitic nematodes of the genus Meloidogyne and investigated their origins, evolution, and characteristics. Using a comparative genomics framework across 85 nematode species, we found that 18% of Meloidogyne genes are genus-specific, transcriptionally supported orphans. By combining ancestral sequence reconstruction and synteny-based approaches, we inferred that 20% of these orphan genes originated through high divergence, while 18% likely emerged de novo. Proteomic and translatomic evidence confirmed the translation of a subset of these genes, and feature analyses revealed distinctive molecular signatures, including shorter length, signal peptide enrichment, and a tendency for extracellular localization. These findings highlight orphan genes as a substantial and previously underexplored component of the Meloidogyne genome, with potential roles in their worldwide parasitism.

Jellyfish have complex life-cycles, but there has been limited exploration of how this is achieved at the cellular level. We used single-cell transcriptomics to assemble a cell atlas for the planula larva of Clytia hemisphaerica, and compared it to an updated cell atlas for the medusa (jellyfish) stage. The cells of the planula fell into the same broad categories as for the medusa: ectoderm, gastroderm, interstitial cells (i-cells), nematocytes (stinging cells), neurons and secretory cells. Although the planula cells generally showed less diversity than medusae within each category, cells with specialized features unique to their stage could be distinguished by their transcriptional profiles as well as by ultrastructure. Some planula-specific types were identified: aboral secretory cells involved in settlement, and a cell type attributed a role in immunity or post-metamorphic theca production. Distinct transcriptome profiles within different regions of the ciliated planula ectoderm reflected different post-metamorphosis fates of domains along the oral-aboral axis. Inspection of the cell clusters showing significant similarity of marker genes between planula and medusa, and inference of similarity using a statistical model of marker gene presence/absence, revealed correspondences between families of cells from planula and medusa rather than precise cell identities.

Trypanosomatid parasites of the genus Leishmania rely on post-transcriptional regulation of gene expression because gene transcription is not canonically regulated at the single-gene level. RNA-binding proteins (RBPs) are central to this regulatory architecture, yet their genome-wide diversity, conservation, and condition-specific associations remain incompletely defined across the genus. Here, we combined comparative genomics and systems-level transcriptomic analyses to map the RBP repertoire in 33 strains spanning 19 Leishmania species. We also connect RBPs to developmental, stress, and drug-resistance contexts, using Leishmania braziliensis as an example. Using probabilistic domain detection and homology-based annotation, we identified 38,662 putative RBPs across all genomes, with 1,114 to 1,491 RBPs per genome. Comparative genomics analysis revealed a conserved core of 404 RBP clusters shared across all examined species, alongside lineage-restricted clusters in major Leishmania groups. We notably detected widespread conservation of enzymes linked to messenger RNA modification (for example, NAT10, TRMT6/61A, and NSUN2), but failed to identify canonical N6-methyladenosine writer orthologs, suggesting divergence of this machinery across Leishmania genomes. In L. braziliensis, expression profiling in different life-cycle stages and stress conditions highlighted stage-biased RBPs, including elevated ZC3H20 in amastigotes and increased RBP6 in metacyclic promastigotes. Finally, co-expression network analysis of trivalent antimony (SbIII) resistant genotypes identified RBPs co-expressed with genes previously associated with antimony resistance. In contrast, motif-based analysis supported a putative DRBD3-centered post-transcriptional module that includes 10 candidate stabilized transcripts in SbIII-resistant promastigotes. Together, these results provide a comparative framework to prioritize RBPs and associated regulatory programs implicated in parasite adaptation and antimony resistance. AUTHOR SUMMARYLeishmaniasis results from infection by Leishmania parasites and remains challenging to control due to limited treatments and the continual emergence of drug resistance, especially to antimonials. These parasites exhibit an unusual gene regulation method; instead of activating or deactivating transcription for individual genes, they depend heavily on proteins that bind RNA to determine which messages are retained, translated, or degraded. In this study, we created a comprehensive atlas of RNA-binding proteins across the Leishmania genus by analyzing 33 parasite strains from 19 species. We identified a large core set of conserved RNA-binding proteins shared by all species, along with lineage-specific proteins that may help different parasite groups adapt to various hosts and environments. Additionally, we mapped enzymes responsible for RNA chemical modifications and found that the typical machinery for m6A marks in many organisms appears to be divergent in Leishmania. Finally, by associating RNA-binding proteins with specific life-cycle stages, stress responses, and antimony resistance in L. braziliensis, we pinpointed candidate regulators and gene modules for further experimental validation. This resource helps prioritize regulatory factors that could drive parasite adaptation and resistance.

BackgroundApproximately one-third of men having undergone gonadotoxic treatment in their childhood experience impaired testicular function for whom autologous transplantation of cryopreserved immature testicular tissue may represent the only opportunity to restore their fertility. Pre-clinical studies have demonstrated successful restoration of spermatogenesis following grafting of immature testicular tissue in various species, including non-human primates. In 2002, our institution pioneered with clinical testicular tissue banking for fertility preservation in boys and adolescents. Over time, this strategy has been increasingly implemented by numerous fertility centres worldwide for patients at high risk of treatment-induced sterility. Here, we report the first human case of autologous transplantation of frozen-thawed immature testicular tissue. PatientIn 2008, testicular tissue was cryopreserved from a pre-pubertal boy diagnosed with sickle cell disease. The procedure was performed after a three-year hydroxyurea treatment and prior to receiving conditioning therapy with busulfan and cyclophosphamide for haematopoietic stem cell transplantation. One testis was surgically removed, sectioned into small fragments, and cryopreserved. Histological analysis confirmed preserved tubular architecture and the presence of spermatogonia. During the period from 2022 to 2024, the patient consistently presented with azoospermia. In December 2024, at the time of transplantation, two abnormal sperm cells were detected after enzymatic digestion. MethodEleven testicular tissue fragments (4-21 mm3) were thawed and autologously grafted to four intra-testicular and four subcutaneous scrotal sites. Over a one-year follow-up period, graft survival, vascularization, hormone profiles, and semen parameters were monitored. One year after transplantation, all grafts were surgically retrieved. ResultsPost-operative recovery was uneventful. No significant changes in endocrine or semen parameters were observed during follow-up. Whereas the intra-testicular grafts exhibited a compact parenchyma that was distinct from the looser surrounding adult parenchyma and remained readily identifiable as graft tissue, the scrotal grafts appeared more fibrotic. Enzymatic digestion of the grafts was required to recover spermatozoa, with one spermatozoon obtained from one of the four intra-testicular grafts. Histological evaluation revealed intact tubular architecture and maturation of somatic cells across all grafts. Spermatogonial stem cells, together with evidence of active spermatogenesis, were identified in two of the four intra-testicular grafts, whereas no germ cells were detected in the subcutaneous scrotal grafts. ConclusionThese findings demonstrate that human immature testicular tissue can survive long-term cryostorage, revascularize after transplantation and establish spermatogenesis in vivo. This study provides essential proof-of-concept for fertility restoration in individuals who banked testicular tissue before puberty. FundingThis study was supported by the Research Programme of FWO Vlaanderen (Research Foundation-Flanders; G0A6U25N) and VUB strategic research program (SRP89). Trial Registration: NCT05414045