BMC Biology

BackgroundHalyomorpha halys (brown marmorated stink bug) is an invasive polyphagous pest causing significant agricultural damage worldwide and is an emerging target for RNAi-based pest management. Despite growing interest in dsRNA-based biocontrol, progress is constrained by the lack of tissue-resolved transcriptomic resources covering key biological processes such as feeding, detoxification, and reproduction. Furthermore, our understanding of how RNAi machinery expression varies across tissues remains limited, which impairs both target gene selection and predictions of RNAi efficacy. Critically, the transcriptional response of H. halys to haemolymph-delivered non-specific dsRNA represents a key knowledge gap for evaluating potential non-target immune reactions of dsRNA-based approaches. ResultsField-collected adult males were injected with either nuclease-free water or dsRNA targeting GFP (dsGFP), and transcriptomes were generated from the brain, midgut, salivary glands, and testes. Sequencing produced high-quality datasets with clear tissue-level separation and tight clustering of biological replicates. As expected in targeting a non-endogenous gene, differential expression analysis revealed a limited transcriptional response to dsGFP. Baseline profiling of RNAi pathway genes in controls showed broad expression of core siRNA and miRNA components across all tissues, yet with marked specialisation: two additional Argonaute-2 isoforms and multiple piRNA factors were testes-specific, whereas salivary glands showed strong, restricted expression of nuclease-encoding genes, including a T2 ribonuclease and a non-specific endonuclease. Expression atlases also revealed pronounced tissue partitioning for other protein families. Consistent with their respective functions, secreted trypsins and chymotrypsins are salivary-enriched while the cathepsins for intracellular protein catabolism are midgut-enriched, with brain-centred neuropeptide expression. However, we also uncovered unexpected nuance, such as closely related subfamilies of Cytochrome P450s, which generally function as detoxification enzymes, being partitioned between the midgut, brain or testes. ConclusionsThis work delivers the first tissue-resolved transcriptomic atlas of adult male H. halys, providing a high-resolution resource on compartmentalization of proteolysis, detoxification, and neuroendocrine signalling, as well as for candidate gene discovery in RNAi-based pest control. The modest, tissue-restricted transcriptional response to non-specific dsRNA, together with strong tissue-specific enrichment of some components, offers mechanistic insight into tissue-dependent RNAi efficiency and supports rational dsRNA target selection in H. halys.

The rat tapeworm, Hymenolepis diminuta, is an important laboratory model for uncovering molecular processes that underly the success of tapeworms as parasites. Despite its importance, a high-quality reference genome for this species is lacking. Here we present a highly contiguous and effectively complete genome of H. diminuta assembled from PacBio HiFi long-read sequencing data. Our primary assembly consists of 7 scaffolds (N50=29.25 Mb) with total length of 186.53 Mb, has only 7 gaps, and contains 95.7% complete Lophotrochozoan BUSCOs. Our assembly allows us to confirm aspects of Hymenolepis genome organization, such as high repeat content and unusual chromosomal ends, and to show that Hymenolepis genomes encode [~]10,000 genes. Together with annotations of nuclear tRNAs, mtDNA protein coding genes, and mtDNA tRNAs, our assembly currently provides one of the most complete genome resources for a tapeworm species and will enable research on parasitism, animal regeneration, development, and evolution.

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.

Microalgae are key primary producers in marine ecosystems, and their interactions with the surrounding environment rely on the secretion of intracellular metabolites and macromolecules, particularly proteins, supporting essential functions such as nutrient acquisition, environmental sensing and biotic interactions. Most abundant and ecologically relevant seawater algae are secondary endosymbionts, where multiple endosymbiotic events extensively reshaped plastids and intracellular membrane systems, requiring adaptation of protein trafficking mechanisms. This study presents the identification of signal peptides that direct protein secretion in the seawater microalga Nannochloropsis oceanica. Their expression in frame with a fluorescent tag enabled to reconstruct the protein secretion pathway in this organism. Proteins channelled for export are first targeted to the periplastidial compartment, an exclusive structure of secondary endosymbiotic algae, that acts as hub for protein trafficking. Subsequently, vesicle-mediated transport directs proteins through the endoplasmic reticulum into the periplasmic space between the cell membrane and the cell wall, from where they are released upon cell division. These findings reveal an evolutionarily remodeled protein secretion pathway, in which host- and endosymbiont-derived trafficking mechanisms merged into an integrated functional system. Significance StatementThe most abundant and ecologically relevant marine algae are secondary endosymbionts whose evolution required extensive re-adaptation of multiple cellular processes. Among them, protein secretion is essential for the interaction with external environment, and required specific re-shaping to the increased cellular complexity associated with endosymbiosis. This work uncovers protein secretory pathway in the secondary endosymbiont seawater alga Nannochloropsis oceanica showing that is does not follow a direct route, but proteins are first accumulated in the periplastidial compartment, a unique structure derived from its endosymbiotic history, before being directed for secretion. The final pathway integrated components derived from both the host and endosymbiont, highlighting how evolution was able to merge different biological modules to build an integrated and functional system.

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.

Non-obstructive azoospermia (NOA) represents the most severe form of male infertility, severely limiting a patient's prospects for biological fatherhood when surgical retrieval fails. However, the true biological limits of NOA remain obscured by the inherent limitations of conventional gamete recovery protocols: standard centrifugation frequently causes substantial cell loss, masking extremely rare sperm, while surgical interventions are constrained by spatial sampling biases. Here we report SpermSeek, an integrated AI-guided microfluidic platform for real-time, non-destructive isolation of single sperm directly from semen. Operating at scalable throughput (0.36 mL/h), the system achieves 98.3% detection precision and a 95.5% target encapsulation efficiency, suppressing background debris. In a 59-patient NOA cohort, SpermSeek detected morphologically identifiable sperm in 64.4% (38/59) of cases, spanning diverse genetic etiologies, including AZFb/c microdeletions, and severe histopathological phenotypes, such as Sertoli-cell-only syndrome (SCOS). Notably, among a sub-cohort of 41 patients who remained consistently sperm-negative despite prior medical or micro-TESE interventions, our platform identified gametes in 53.7% (22/41) of these cases. Comprehensive safety profiling in healthy human donors and wild-type mice confirmed that processed sperm retain high DNA integrity and epigenomic concordance (r=0.98), supporting transgenerational developmental stability in mice. Furthermore, in a 26-patient validation cohort, SpermSeek recovered rare sperm in 11 cases. Utilizing gametes from a subset (n=5), we demonstrated their capacity to support early human embryogenesis, yielding high-quality cleavage-stage embryos with confirmed genomic euploidy. This work establishes a highly sensitive framework for re-examining the biological limits of human spermatogenesis, laying the foundation to expand autologous reproductive options for patients refractory to conventional retrieval protocols.

Coleoid cephalopods, squids, cuttlefish, and octopuses, have emerged as powerful model organisms for studying neurobiology, development, and behavior, however, cellular tools for investigating their specialized tissues remain limited. In particular, their venom producing gland, the posterior salivary gland (PSG), has been extensively described anatomically and histologically, yet remains largely inaccessible to experimental investigation at the cellular level. Here, we report the first establishment of primary cell cultures derived from both the optical lobe and PSG tissues of Octopus bimaculoides. Building on recent advances in cephalopod brain cultures, we adapted and optimized dissociation and culture conditions to support short-term survival and attachment of cells in vitro. We show that passive cell release during tissue handling, rather than enzymatic treatment, yields viable cultures from both tissues, and poly-D-lysine markedly improves the adherence of PSG-derived cells. Morphological analyses and fluorescent staining confirm the presence and viability of distinct cell populations, while cell cycle analysis indicates that the majority of cells reside in G0/G1 phase. Notably, O. bimaculoides brain cultures exhibit features comparable to those previously described in squid, suggesting conserved aspects of coleoid cellular physiology. Together, our findings establish a foundational in vitro platform for studying octopus PSG and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods.

Among venomous animals, cnidarians represent the oldest metazoan lineage in which venom production and a specialized delivery system are defining synapomorphies. Cnidarians also represent the only venomous lineage for which mutualistic symbioses have evolved resulting in scenarios where mutualistic symbionts may also be targets of their venom. The most iconic example of this relationship is the mutualism between clownfish and their venomous sea anemone hosts. To investigate how symbiont presence and establishment influence toxin gene expression, we used a comparative TagSeq and RNA-Seq approach to quantify venom gene dynamics during the first 48 hours of clownfish-anemone symbiosis establishment in five anemone species. Our taxonomic sampling included three typical hosting species (Entacmaea quadricolor, Radianthus crispa, and Stichodactyla haddoni), each representing distinct evolutionary lineages of clownfish hosts, and two atypical Caribbean species (Condylactis gigantea and Stichodactyla helianthus) that do not host clownfish in nature, but have reported to host within the aquarium trade. Tentacle samples were collected prior to hosting, approximately 12 hours after initial symbiont establishment, and again 48 hours after symbiosis establishment. Our analyses revealed that overall toxin assemblages remained relatively stable during the early establishment phase, with no significant changes in the most highly expressed toxin gene candidates. However, subtle transcript-level shifts occurred within multi-copy toxin gene families, including cytolytic actinoporins and Sea Anemone 8 (SA8)-like toxins. Notably, one C. gigantea actinoporin transcript exhibited a [~]600-fold increase in expression in a single individual, which coincided with two clownfish mortalities prior to successful association, which subsequently decreased after establishment. Comparative sequence alignments suggest that amino acid substitutions in this transcript may be functionally relevant to symbiosis intolerance, as the amino acid substitutions were unique to this transcript, and not found in any other previously described cytolytic actinoporin. Together, these findings reveal that early toxin gene expression in clownfish-hosting sea anemones is largely stable, yet subtly dynamic at the transcript level. This study provides the first comparative transcriptomic insights into the molecular processes shaping symbiosis establishment in clownfish-anemone mutualisms, offering a framework for understanding venom evolution in the context of co-evolutionary interactions. HighlightsO_LIComparative gene expression survey reveals relatively stable toxin assemblages throughout the first 48 hours of establishing clownfish-anemone symbiosis. C_LIO_LISubtle shifts were observed among transcript variants in multi-gene copy variants, with potential implications for barriers to establishing symbiosis. C_LIO_LIAlthough toxin assemblages varied among species, sea anemone 8 (SA8) toxin-like transcripts were highly abundant in four of the focal taxa. C_LIO_LIThis is the first comparative gene expression analysis investigating molecular processes surrounding symbiosis establishment between clownfish and sea anemones. C_LIO_LIThese results provide insight into toxin dynamics surrounding the establishment of symbiosis, with particular insights into key evolutionary transitions resulting in symbiosis among atypical clownfish hosting species. C_LI

The phylum Mollusca constitutes one of the most taxonomically and morphologically diverse animal clades; however, the genomic exploration of this group has been hampered by fragmented and taxonomically incomplete transcriptomic resources. To address this fundamental limitation, we present MolluscaGenes, a centralized database that unifies transcriptomes from 299 molluscan species spanning all eight recognized classes, encompassing a broad array of tissues and developmental stages. MolluscaGenes provides searchable databases via BLAST and DIAMOND alongside a suite of 196 molluscan-optimized Hidden Markov Models (HMMs) for sensitive protein family identification. To demonstrate the utility of this resource, we performed a comprehensive phylum-wide characterization of the nicotinic acetylcholine receptor (nAChR) superfamily, recovering 3,586 sequences from over 190 species and resolving 15 distinct phylogenetic clades. This analysis revealed substantial lineage-specific expansions across multiple molluscan classes, the identification of novel clades with substitutions in canonical ligand-binding residues, and the evolutionary placement of chemotactile receptors (CRs) and CR-like sequences as predominantly cephalopod clades within the broader nAChR phylogeny. MolluscaGenes constitutes a foundational resource that will accelerate the elucidation of the unique biology and evolutionary history of Mollusca.

The population of Madagascar exhibits a globally unique combination of African and Asian genetic ancestries. Previous studies have described the admixture history of Madagascar at island-wide scales [1,2], but less focus has been paid to fine-scale population structure across the island. We present new genome-wide genetic data from 192 individuals sampled across five regions of Madagascar. We identify population structure at extremely fine spatial scales ([~]10 km) among the Merina of the central highlands. By analysing subpopulations separately, we found one Merina group exhibited similarity to coastal populations in f4 ratios, estimated admixture dates, and pairwise FST distances, while another group was similar to other highland individuals in the same measures. This fine-scale substructure is likely associated with historical coastal-to-highland migration during the 18th and 19th centuries. In contrast, we also observe macro-scale structure in estimated timing of admixture across the island, with southeastern coastal groups exhibiting the earliest estimated admixture timings, and northern groups exhibiting the latest. This pattern corroborates previous results [1,2], and may suggest differing histories of admixture timing among Malagasy populations. Our results emphasise the importance of deep micro-geographic sampling to complement macro-scale analysis when characterising demographic history.

Cephalopod arms are controlled by a distributed peripheral nervous system comprising the axial nerve cord (ANC), subacetabular ganglia associated with each sucker, four longitudinal intramuscular nerve cords (INCs) embedded within the arm musculature and oblique connectives (OCs) running between INCs. Despite their prominent anatomical position and proposed roles in local sensorimotor integration and inter-arm coordination, the INCs remain poorly characterized with respect to cell-type composition and molecular identity. Here, we report the first integrative characterization of INC structure and composition in Octopus bocki by combining serial block-face scanning electron microscopy (SBEM) with multiplexed hybridization chain reaction (HCR) in situ labeling. We show that oral and aboral INCs share a consistent internal organization comprising distinct cell body regions, a peripheral tract layer, and a central synaptic zone. Both oral and aboral cords contain morphologically diverse cell populations, including abundant bipolar neurons with long unbranched processes and a second class of neurons with highly branched processes bearing bouton-like enlargements. On the molecular level, the sampled INCs are enriched for glutamatergic and buccalin-positive cells, and express abundant glia-associated transcripts. In contrast to the ANC, cholinergic, dopaminergic, serotonergic, and octopaminergic markers were not detected above background. We also characterize the relationship between the INCs and adjacent oblique connectives (OCs), showing that these structures run in close proximity but remain physically separate within the sampled high-resolution volume, with no shared fibers or crossing processes detected across the observed boundary. Together, these data establish a cellular and molecular framework for the INCs and clarify their relationship to neighboring peripheral pathways.

Conserved dorsoventral patterning systems have been proposed as evidence for a common evolutionary origin of centralized nervous systems in Bilateria, yet functional evidence outside vertebrates and arthropods remains limited. Here, we investigated the role of pax6 in the annelid Platynereis dumerilii using a mutant carrying a 61 bp deletion in the paired-domain coding region. Loss of pax6 disrupted ventral neuroectodermal patterning at 34 hpf, causing a shift in nk2.2 expression, narrowing of the nk6 domain, and downregulation of pax3/7, while msx expression remained largely unaffected. These early patterning defects were followed by selective neuronal abnormalities at 48 hpf, including displacement of TrpH-positive serotonergic cells and loss of posterior hb9-positive motoneuron domains. By 6 dpf, additional defects were observed in TrpH, ChAT, VAChT, and nk2.2 expression, accompanied by severe disruption of ventral nerve cord morphology and loss of the characteristic rope-ladder architecture. Together, these findings identify pax6 as a key regulator linking dorsoventral progenitor patterning, neuronal subtype specification, and nervous system morphogenesis in Platynereis. Our results provide functional evidence that the conserved dorsoventral patterning network plays an essential role in annelid ventral nerve cord development and support the view that important components of bilaterian nervous system patterning predate the divergence of major animal lineages.

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

Animal venoms represent a major source of chemical novelty, yet how venom compounds originate, diversify, and are maintained across deep evolutionary timescales remains poorly understood. This gap is especially pronounced in cephalopods, which evolved venom systems used in predation, defense, and sexual competition, but whose venom genetic architectures, secretory cell types, and venom-producing glands remain largely unexplored. To date, only a single cephalopod venom compound with confirmed paralytic activity and a known primary sequence, SE-CTX from the golden cuttlefish Acanthosepion esculentum, has been described. Here, we reconstruct the evolutionary history, molecular diversity, and glandular localization of SE-CTX-like proteins using a multimodal approach. We identify 29 homologs across 20 squid and cuttlefish species and define a previously unrecognized venom gene family, which we name deca-ctx, specific to decapodiform cephalopods (squids and cuttlefish). Phylogenetic analyses reveal a single origin of deca-ctx followed by gene duplication and lineage-specific diversification, indicating long-term retention of this venom gene. Predicted DECA-CTX protein structures were separated into two clusters and 20 singletons highlighting potentially extensive structural diversity within a single cephalopod venom gene family. Proteomic analysis confirms expression of five DECA-CTX proteins across three species. Our imaging and histological analyses localize deca-ctx expression to specialized secretory cells within squid and cuttlefish venom glands. Together, these findings reposition SE-CTX as part of an evolutionarily and chemically diverse venom system, rather than an isolated venom protein, and establish cephalopods as a key lineage for investigating how new venom genes arise, diversify, and are integrated into functional venom arsenals.

TULIP (TUbular LIPid binding) domain proteins (TDPs) are found in all living organisms including bacteria. They have various documented functions, some of which clearly related to their intra- or extracellular lipid transfer activities. Extracellular, BPI-related TDPs of insects (B-TDPs, also known as Takeout-related proteins), are often found in chemosensory organs, but little is known regarding their exact location or how they could contribute to olfaction or gustation. We have surveyed and updated the full set of Drosophila B-TDPs and found that roughly 50% are overexpressed in chemosensory organs. Focusing on three genes clustered on the third chromosome, we provide evidence that at least one of the encoded proteins is secreted in the lymph cavity housing the dendrites of olfactory neurons. Biochemical data give support for a putative function of B-TDPs as odorant transporters, but loss-of-function analyses also hint to a potential role as a barrier against plant-emitted terpenoids.

In trypanosmatids genes, transcribed by RNA polymerase II do not have canonical promoters and are organized into directional gene clusters that mature into monocistronic transcripts by a co-transcriptional process known as trans-splicing. Even though gene expression is regulated mainly post-transcriptionally, it is currently understood that chromatin and epigenetics are also involved in this regulation. In eukaryotes, specific signals are normally required for the occurrence of an appropriate transcription initiation. Among them, trimethylation of histone H3 in lysine 4 is the most conserved signal normally detected at transcription start sites of actively transcribed genes. Unlike many model organisms, trypanosomes do not have defined promoters. Instead, transcription initiates in a bidirectional manner from dispersed regions coincident with divergent strand switch regions located between directional gene clusters (DGCs). In T. cruzi, H3K4me3 was observed at the origins of transcription coincident with divergent strand switch regions (dSSRs) in epimastigotes, but it has not been mapped throughout the whole genome at base-pair resolution or in other life stages so far. Here, we set up the CUT&RUN technique for T. cruzi epimastigotes and trypomastigotes. Consistent with a predominant post-transcriptional regulation along the life cycle, we did not find significant differences between life stages. We corroborated that H3K4me3 is enriched at dSSR adjacent to actively expressed DGCs. Moreover, we noticed that this histone mark exhibits different patterns that correlate with the genomic span of the transcription initiation regions and with transcriptional activity. Furthermore, we unveiled that the most actively transcribed DGCs are associated with shorter dSSRs and are located within the core compartment of the genome displaying a more accessible chromatin.

Evolutionary success in mammals requires coordinated regulation of cognitive functions and reproductive capacity. Such coordination must involve shared genes and molecular pathways between the brain and germ cells, yet direct evidence linking cognition to reproduction across species remains limited. Here, proteomic and transcriptomic analyses were performed experimentally in Ovis aries and Rattus norvegicus, while transcriptomic datasets from Mus musculus, Macaca mulatta, and Homo sapiens were analyzed in silico. We identified 8,464 protein-coding genes shared between the brain and sperm/testis and conserved across five species. In rats, 8,444 of these genes were also shared between the brain and the ovary. Functional annotation classified 3,890 genes as associated with both neurological and reproductive functions, and 1,752 as uncharacterized in these contexts, highlighting candidates for future studies on reproductive and neurological disorders. These findings reveal a deeply conserved genetic network linking neurological and reproductive systems, underscoring the evolutionary interplay that supports mammalian fitness.

Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H-ATPase (V-H+-ATPase), Ca{superscript 2}-transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites. Author summaryProtozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases.

Immunoglobulin genes are a central component of jawed-vertebrate adaptive immunity. A previous study showed that the blunt-snouted clingfish Gouania willdenowi lacks immunoglobulin genes and T-cell receptor gamma/delta loci, while retaining T-cell receptor alpha/beta genes, MHC genes, and RAG1 /RAG2. Here I extend that observation to the family Gobiesocidae using all seven chromosome-level Gobiesocidae genome assemblies currently available. Manual tblastn and synteny-guided searches found no convincing immunoglobulin heavy-chain or light-chain loci in G. willdenowi, Gouania pigra, Gobiesox punctulatus, Apletodon dentatus, Lepadogaster candolii, Lepadogaster purpurea, or Diplecogaster bimaculata. Thus, the absence of antibody genes is best interpreted as a root-level character of clingfishes. The latest seven-species screen of 40 additional immune-associated genes shifts the broader interpretation in the same direction: the B-cell/adaptive core genes CD79A, CD79B, CIITA, TNFRSF13B, and TNFSF13B lack strong tblastn support in all sampled Gobiesocidae, and 37 of the 40 tested targets show an all-zero binary pattern at the presence threshold. Only IL21R.1, TYROBP, and TNFRSF11A show strong hits in one or more species. I therefore interpret the principal immune-gene erosion as occurring at or near the Gobiesocidae root rather than as a recent Gouania-specific process, while keeping weak, paralog-sensitive, and patchy loci provisional. RAG2 comparisons show a shared Gobiesocidae PHD-domain C-to-S replacement in the zinc-binding motif, with apparently intact RAG2 coding sequence. A family-wide TRG/TRD screen did not recover TRGV V segments or accepted TRDC constant-region exons, but it did detect TRGC-like constant exons in several genomes. These TRGC-like sequences are probably not canonical TRG constant exons without further validation, so I treat the gamma/delta system as eroded or rearranged rather than as a complete root-level loss equivalent to the Ig loss. The RAG2 variant provides a plausible molecular context for antigen-receptor remodeling, but it is not evidence that RAG genes are pseudogenized, because TCR alpha/beta, MHC genes, and RAG1 /RAG2 are retained. Gobiesocidae are therefore best described as a vertebrate family with ancestral loss of canonical immunoglobulin genes and associated root-level erosion of B-cell and immune-related genes, not as a lineage lacking adaptive immunity in its entirety. HighlightsO_LISeven chromosome-level Gobiesocidae genomes lack convincing canonical IgH and IgL loci. C_LIO_LIThe strongest non-Ig losses map to the B-cell/adaptive core: CD79A, CD79B, CIITA, TNFRSF13B, and TNFSF13B. C_LIO_LITCR alpha/beta, MHC genes, and RAG1 /RAG2 are retained, so Gobiesocidae should not be described as lacking adaptive immunity in full. C_LIO_LIA shared Gobiesocidae RAG2 PHD-domain C-to-S variant provides candidate molecular context for antigen-receptor remodeling. C_LI

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG