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.

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.

During meiosis, homologous chromosomes pair to form synaptonemal complexes (SCs) and exchange genetic material through a process known as meiotic recombination. First, programmed DNA double-strand breaks form, followed by the assembly of recombination foci on SCs. These foci mark the sites of recombination intermediates and future crossovers. Distributions of recombination foci along SCs have been studied in many eukaryotes, revealing the interplay between recombination patterns and genome evolution. However, in fish, data on recombination patterns are scarce, and, for the majority of groups, completely absent. Here, we measure the positions of MLH1 foci in 3,504 SCs from 219 male meiotic cells of an African annual killifish Nothobranchius virgatus, a representative of a genus with remarkable karyotype and genome diversity, and present a detailed statistical analysis of its recombination patterns. We found that, in contrast to the several other fish species characterised to date, recombination in N. virgatus occurs across almost entire chromosome arms, excluding (peri)centromeres and telomeres. In the longest SCs, we observed a proximal and a distal peak of the recombination focus frequency and explained the peaks by chromosome pairing dynamics. We also revealed the typical positions of focus pairs, demonstrated interference between foci, with the minimal interfocus distance of 4 m, and described regions of the total recombination suppression near centromeres and telomeres. In sum, our study provides a detailed analysis of recombination patterns in a killifish with a fully acrocentric karyotype and contributes to cytogenomic and statistical methodology for future exploration of meiotic recombination patterns.

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.

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.

We report the identification and functional validation of a 7SK RNA polymerase III promoter in the Mediterranean fruit fly, Ceratitis capitata. CRISPR/Cas9-based genetic control strategies for this global agricultural pest, including gene drives and precision guided sterile insect approaches, require efficient guide RNA expression, yet only a single U6 Pol III promoter had previously been validated for this purpose in C. capitata, and no 7SK promoter had been characterised in any Tephritid species. Using comparative genomics with Drosophila orthologues, we identified a previously unannotated 7SK gene in the C. capitata genome, confirmed its transcriptional activity by RT-PCR, and demonstrated that the cloned promoter drives functional guide RNA expression in CRISPR/Cas9-mediated knockouts of the white gene. Comparative analysis identified putative 7SK orthologues across the Tephritid fruit flies. The availability of this additional new Pol III promoter will enable multiplexed guide RNA strategies using distinct promoters, supporting more robust genetic control designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719894v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@156c203org.highwire.dtl.DTLVardef@db5eedorg.highwire.dtl.DTLVardef@353adforg.highwire.dtl.DTLVardef@ac079a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Triatomines are the vectors of Chagas disease, one of the main endemic diseases from South to North America, now expanding to other continents. These hemimetabolous insects have been considered poorly visual animals. However, recent findings challenge this idea. Here, we used Rhodnius prolixus as a model species to comprehensively characterize triatomine compound eyes. We found that in the adult stage, eye size significantly exceeds the dimensions predicted by the nymphal eye growth rate. Moreover, while the compound eye grows symmetrically in its dorsal and ventral directions throughout the nymphal instars, in the adult, the eye undergoes greater ventral growth, resulting in a dorsoventrally asymmetrical eye. By studying a bright pseudopupil induced by fluorescence in natural mutant animals, we observed no major differences in sampling resolution between the last nymphal instar and the adult stage. However, the adult eye possesses significantly larger ommatidia, particularly in its ventral region, shifting the area of highest sensitivity from the equatorial region in the nymphal instars to the ventral region in the adult. A similar eye growth pattern was observed in Triatoma infestans and Panstrongylus megistus. The analysis of photographic records from 39 species across 10 genera indicates that an asymmetrical eye is the predominant eye pattern in adult triatomines. Notable exceptions in wingless adults of Mepraia spinolai, reveal a tight association between possessing a large asymmetrical eye and the presence of wings. This suggests that vision might support triatomine dispersal flights among other visual behaviors. Significance StatementKissing bugs are hematophagous insects known for being the vectors of Chagas disease, one of the main endemic diseases in the Americas. Vision was not considered a relevant sensory system in these insects. Here, we show that their eyes increase in size beyond expected by ontogeny and become asymmetrical when transitioning from the last nymphal instar to the adult stage. The eyes undergo a ventral expansion that shifts the region of greatest light sensitivity from the equatorial zone in nymphs to the ventral region in adults. We found this asymmetrical eye only in winged kissing bugs, suggesting that vision supports flight. This is relevant in ecological and epidemiological terms since kissing bugs disperse by flight for habitat colonization and host-seeking.

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

Cleptoparasitism, or brood parasitism, is a striking behavioral strategy observed in approximately 13% of all bee species, yet its genomic underpinnings remain largely unexplored. We present the first high-quality genome assembly of the Neotropical cleptoparasitic bee Coelioxoides waltheriae (Nomadinae), a species that parasitizes the nests of Tetrapedia diversipes. The final assembly comprises 194.8 Mbp across 388 contigs, with an N50 of 1.47 Mbp and 97.4% BUSCO completeness, representing the second smallest genome among cleptoparasitic bees. Repetitive elements constitute only 14.6% of the genome, suggesting that its compact size is primarily driven by repeat reduction rather than gene loss. Comparative genomic analyses across 42 hymenopteran species revealed a pronounced contraction bias in gene family size changes in C. waltheriae (expansion ratio of 13.66%), a pattern also observed in other cleptoparasitic lineages. Expanded orthogroups were enriched for cuticle-related genes (e.g., PiggyBac transposases) potentially linked to host infiltration and defense, while contracted orthogroups showed significant reductions in sensory perception (e.g., odorant receptors), detoxification (e.g., cytochrome P450), and metabolic genes, reflecting the reduced ecological demands of a parasitic lifestyle. Furthermore, non-target DNA analysis identified associations with Roubikia mites (a known symbiont of its host), as well as fungi and bacteria, providing ecological context for this species. Our findings establish a critical genomic reference for cleptoparasitic bees, demonstrating that the evolution of parasitism is associated with targeted gene family contractions in sensory and metabolic functions alongside expansions in cuticle and transposable element-related genes, offering new insights into the genomic signatures of behavioral specialization.

STUDY QUESTIONAre pathogenic variants in Homeodomain-interacting protein kinase (HIPK4) associated with sperm head abnormalities causing male infertility? SUMMARY ANSWERHIPK4 is a novel candidate gene associated with sperm head defects and human male infertility. WHAT IS KNOWN ALREADYNumerous genes causing male infertility due to Multiple Morphological Abnormalities of the sperm flagella (MMAF) have been described but the genetic basis of sperm head defects is less well understood. STUDY DESIGN, SIZE, DURATIONFour infertile brothers displaying varying degrees of quantitatively and/or qualitatively impaired spermatogenesis, their parents, and their fertile brother were included in the study. Further, the Male Reproductive Genomics (MERGE) cohort comprising exome/genome sequencing data of >3,300 men was queried. PARTICIPANTS/MATERIALS, SETTING, METHODSWe performed exome sequencing in all five brothers and their parents. To characterise the sperm phenotype, standard semen analysis, immunofluorescence staining, and transmission-electron microscopy (TEM) were carried out. Further, we evaluated the impact of the HIPK4 variant in cell culture experiments using HEK293T cells. MAIN RESULTS AND THE ROLE OF CHANCEAnalysing the exome data, we could not identify a common genetic cause in all four affected brothers. However, one of the affected brothers was compound heterozygous for two loss-of-function variants in DNAH17 (c.1076_1077dup p.(Lys360*) and c.7752+2T>A p.?) associated with markedly reduced sperm motility and MMAF. The variants pathogenicity was further validated by TEM of flagellar cross-sections revealing an outer dynein arm defect and axonemal disruption. On the contrary, his three infertile brothers were homozygous for the start-loss variant c.1A>G in HIPK4. This gene is expressed during spermiogenesis and is reportedly involved in sperm head shaping in mice. Heterologous expression of (partial) HIPK4 variant cDNA elucidated the alternative use of an in frame start codon located 35 amino acids downstream, resulting in an N-terminally truncated protein p.(Met1_Glu35del). The truncated HIPK4 protein lacks parts of its kinase domain and shows reduced protein stability. In line with published mouse models, all three brothers displayed 100% abnormal sperm head morphology with variable defects. Importantly, one brother affected by HIPK4 variants fathered a child after successful intracytoplasmic sperm injection demonstrating that it is a treatment option for HIPK4-related teratozoospermia. No further men from the MERGE cohort were affected by biallelic HIPK4 variants. Taken together, HIPK4 is an autosomal-recessive candidate gene associated with sperm head defects and male infertility. LARGE SCALE DATAThe reported variants in DNAH17 and HIPK4 were submitted to ClinVar. LIMITATIONS, REASONS FOR CAUTIONIndependent replication is required to assess the phenotypic spectrum and the reproductive outcome associated with biallelic HIPK4 variants and to formally establish the gene-disease relationship for male infertility. WIDER IMPLICATIONS OF THE FINDINGSThis study raises awareness of the significant genetic heterogeneity of male infertility. The described family highlights that distinct genetic causes may underlie a seemingly similar phenotype. Exome sequencing of families is helpful to efficiently disentangle individual causes among affected family members. STUDY FUNDING/COMPETING INTEREST(S)N.N., J.R., H.O., S.L., C.F., and F.T. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit Male Germ Cells (CRU326, project number 329621271). R.T.W., N.N., J.R., H.O., and F.T. were supported by the Federal Ministry of Research, Technology and Space (BMFTR) as part of the project ReproTrack.MS (grant 01GR2303). S.A.K. was supported by the DFG Clinician Scientist programme CareerS Munster (project number 493624047). A.S.G. was supported by the Medical Faculty Munster via an Innovative Medical Research (IMF) grant (GA-122104).

Following the evolution of internal fertilisation, the female reproductive tract became the site of reproductive interactions. However, our understanding of the evolution of female reproductive tract function, including postmating responses critical for reproductive success, are taxonomically limited. Traumatic insemination in the common bedbug (Cimex lectularius) presents an unusual scenario under which postmating responses unfold. Bedbugs have evolved a novel organ, the mesospermalege, that is the site of initial ejaculate x female interactions. As the female reproductive tract does not take receipt of the ejaculate until several hours after mating, bedbugs provide a unique opportunity to explore the evolution of a novel reproductive organ that decouples postmating female responses involved in mating and transfer of the ejaculate from sperm storage, ovulation, and oviposition. Here we show that the mesospermalege has a gene expression profile consistent with functions of ejaculate processing and immune response normally found in the lower reproductive tract of other insect species. In parallel, the postmating response in the lower female reproductive tract is delayed, coinciding with movement of sperm through the female, clearly showing that the postmating response has evolved in response to sperm receipt rather than being an innate function of the tissue. Notably, we also found expression of male seminal fluid genes in the mesospermalege, indicating that intersexual molecular dynamics influence the evolution of reproductive tissues. Our results provide insights into the evolution of novel reproductive traits and female postmating physiology in a global pest with an unusual reproductive biology. SIGNIFICANCEReproduction poses one of the most persistent challenges faced by animals whereby females undergo a series of physiological changes after mating. The independent origin of a reproductive organ in bedbugs (called the mesospermalege) which has evolved to alleviate the costs of traumatic insemination presents a unique case to study the evolution of a novel trait and postmating physiology. Using transcriptomics, we show that many genes normally expressed in the female reproductive tract are instead expressed in the mesospermalege. The reproductive tract also shows a delayed postmating transcriptional response coinciding with sperm entry into the reproductive tract. Our results provide insights into the evolution of reproductive traits and female postmating physiology in a global pest with an unusual reproductive biology.

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

Secretion signals are ancient and functionally conserved sequence motifs that orchestrate function and intended destination of cleaved encoded proteins (1-3). To investigate the genomic landscape of secreted proteins, 4,694 annotated eukaryotic genome assemblies were analysed. Genes encoding secretion signals (n = 5.2 million) were consistently enriched in genomic regions with longer flanking intergenic regions (FIRs). Consecutive genes with characteristic FIR lengths were enriched for genes with secretion signals. Intriguingly, many eukaryotic pathogens and parasites have the most significant association between genes encoding secretion signals and their intergenic distance. Almost every category of repeat was found in greater number flanking genes encoding secretion signals, with especially strong enrichment of simple, unknown, and low complexity repeats in fungal genomes. Despite higher repeat counts, the total repeat length was consistently shorter around genes with secretion signals, suggesting a prevalence of truncated or fragmented repeats in these regions. Several GO-terms assigned to genes with secretion signals were consistently enriched across genome assemblies in each kingdom. Common GO-enrichment patterns were also identified in genes categorised by their FIR. These results hint at an anciently conserved genomic architecture and mode of evolution in eukaryotes, characterised by long FIRs and fragmented repeat landscapes, likely driven by mechanisms such as repeat-driven gene copy number variation (4), differential mutation rates (5) and chromatin remodelling (6). This conserved association highlights the potential of genome structure to drive innovation in secreted protein function.

The coastal rock pool mosquito, Aedes togoi, is among the few saline-tolerant mosquito species who lay their eggs in seawater pools where their larvae develop in water that spans dilute freshwater to hypersaline conditions. Ae. togoi is found in a relatively restricted range spanning the North Pacific coast of North America and coastal regions of Asia from subtropical to subarctic latitudes. Here, we present a de-novo chromosome-scale genome assembly and gene annotation for Ae. togoi, highlighting its relatively small genome size and novel chromosomal arrangements compared to other available genomes of Aedine mosquitoes. As part of the annotation process, we detail repeat content and distribution and curate several key multi-gene families, focusing on ion-transport proteins enriched in the larval salt-secreting gland that are candidates for facilitating hyperosmotic urine formation during development in saline water. Using these new resources, we gain mechanistic insight into the ion regulatory capabilities that power the remarkable saline tolerance of the larvae of Ae. togoi. Altogether, we have contributed to the growing body of genomic and transcriptomic resources for diverse mosquito species and provided mechanistic insights into the molecular adaptations required for an insect to thrive in highly dynamic environments such as coastal rock pools.