BMC Biology

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.

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.

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.

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.

Accurate genome annotation remains challenging as assembly quality often exceeds annotation reliability. Resolving ambiguities of gene presence, absence, and orthology typically requires integrating two complementary lines of evidence: sequence homology between species and the conservation of gene order (i.e., synteny). BLAST remains the standard for homology detection, yet its raw output can be difficult to interpret. Existing tools address this challenge but operate at opposing scales. Alignment viewers provide detailed pairwise statistics without genomic context, while synteny tools offer chromosome-scale perspectives without sequence-level resolution. To fill this intermediate gap, we developed Novabrowse, an interactive BLAST results interpretation framework featuring high-resolution multi-species synteny analysis, chromosomal re-arrangement investigation, ortholog detection, and gene signal discovery. Users define a genomic region of interest in a query species and/or use custom sequences, then select one or more subject species for comparison. The pipeline retrieves query gene sequences via NCBI API integration and performs BLAST searches against each subject transcriptome or genome. Results are presented via an interactive HTML file featuring alignment statistics, chromosomal maps, coverage visualizations, ribbon plots, and distance-based clustering of high-scoring segment pairs into putative gene units. We demonstrate these capabilities by investigating Foxp3, Aire, and Rbl1, three highly conserved vertebrate genes, in the recently assembled genome of the newt Pleurodeles waltl. Foxp3 and Aire have not been described in any salamander species to date, despite availability of multiple assemblies and extensive transcriptomic datasets. Using Novabrowse, we discovered conserved loci and gene signals for both genes in P. waltl, the presence of which was subsequently confirmed via Nanopore long-read RNA sequencing. In contrast, Rbl1 analysis uncovered a chromosomal rearrangement at its expected locus with no gene signal detected, indicating a gene loss specific to P. waltl despite the genes retention in the closely related axolotl (Ambystoma mexicanum). Our findings demonstrate Novabrowses capacity for evidence-based evaluation of annotation artifacts, an essential capability as high-quality assemblies become more available for phylogenetically diverse species. Novabrowse is open source (MIT license) and freely available at: https://github.com/RegenImm-Lab/Novabrowse.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

tRNA are adapter molecules with an integral role in translation and further roles in stress adaptation. Processing of tRNA is tightly regulated and includes the enzymatic addition of several post-transcriptional modifications that are required for translation efficiency, recognition, selective translation, and structure. We currently lack a multi-species wide view of tRNA modifying enzymes across eukaryotes. Here, we performed a comparative analysis of tRNA gene sequence, modification enzymes, and modification profiles across the Saccharomycotina subphylum. We employed machine learning methods to explore tRNA sequence conservation and to annotate modifying enzymes known to exist in fungi, humans, and prokaryotes. We then applied Nano-tRNAseq to three species (Saccharomyces cerevisiae, Hanseniaspora uvarum, and Yarrowia lipolytica) to profile modification signatures and compare modification patterns. We identified substantial lineage-specific conservation of tRNA sequences despite the highly conserved tRNA structure. We found significant variation in tRNA modifying enzyme repertoires across Saccharomycotina, including lineage-specific losses, and annotated a prokaryotic-associated enzyme, tilS. Integrating genomic and sequencing data enabled us to link enzyme repertoires with tRNA gene sequences. tRNA sequencing revealed distinct modification signatures across the three focal species, and further analysis using General Linearized modelling suggested tRNA enzyme loss is associated with target tRNA nucleotide absence in gene sequences. This work provides the first integrated view of tRNA gene and modification diversity in eukaryotes and expands the field of tRNA diversity in fungi.

Gene drives are genetic elements that bias their own inheritance to spread desired traits in target populations, enabling population modification or suppression. Although homing-based drives can propagate efficiently, their potential for uncontrolled spread may present a challenge for field deployment. Thus, confined drive systems are needed. Here, we developed a confined modification drive, called Toxin-Antidote Recessive Embryo (TARE) drive, in the globally important malaria vector Anopheles stephensi. This drive works by cleaving and disrupting wild-type alleles in the germline or early embryo from maternally deposited Cas9. Disrupted alleles are recessive lethal, thus increasing the drive in a frequency-dependent manner. Inheritance bias was moderate in crosses between drive heterozygote mosquitoes, possibly due to low gRNA activity and thus moderate germline cleavage rates. Single-release cage trials confirmed the TARE drives ability to spread, although the drive ultimately declined due to fitness costs and resistance alleles associated with repetitive elements. Nonetheless our modelling analysis indicate that this TARE system could achieve population spread if the resistance issue is addressed. These findings demonstrate a functional prototype TARE drive in Anopheles stephensi and highlight key parameters governing its performance. Minor design optimizations could substantially improve efficiency and integrity, enabling rapid but confined population modification.

BackgroundAlthough strict maternal transmission of mitochondria is a general feature of animals and humans for ensuring homogeneity in mitochondrial DNA (mtDNA) across generations, exceptions were reported in the recent past. For example, some extremely rare but spectacular cases of heteroplasmy and paternal transmission in humans have questioned the universal evolutionary principle. Hence, as an alternative, the Mega-NUMT concept was coined to explain this discovery and was thereafter partly proven to exist. This concept expands on the quite common transfer of mtDNA fragments to the nucleus (NUMTs) by considering the existence of multicopy mitochondrial nuclear insertions. Mega-NUMT reports are currently restricted to a few cases in animals, including humans. However, even in humans, their detailed genomic organization, natural prevalence, and potential biological functions remain unclear. Methodology/Principal FindingsHere, we discovered that up to 60 full-sized mitochondrial genomes are integrated into the nuclear genome of the neotropical fruit fly Drosophila paulistorum using long-read sequencing and confirmed their presence by in situ hybridization. The copies are organized in one cluster on chromosome 3, which we, due to its similarity with the Mega-NUMT concept, designated the "Dpau Mega-NUMT". Contrary to the rarity in humans, this Mega-NUMT is found at high prevalence (40%) in both long-term laboratory lines and natural D. paulistorum populations of different semispecies. Additionally, the mitochondrial copies in the Mega-NUMT cluster are phylogenetically separated from the current mitotypes of D. paulistorum. Together, these observations suggest long-term maintenance of the Mega-NUMT in nature. Hence, we propose that the Dpau Mega-NUMT may have been transferred to the nuclear genome before D. paulistorum semispecies radiation and maintained at relatively high prevalence in nature by balancing selection due to yet undetermined functions. Conclusions/SignificanceTo our knowledge, this is the first verified existence and detailed dissection of a Mega-NUMT outside cats and humans. We show that Mega-NUMTs can be persistent in nature, even at high prevalence, potentially due to balancing selection. Our findings strengthen the importance of high-quality long-read sequencing technologies for deciphering complex repeat-rich genomic regions to deepen our understanding of the dynamics of genome evolution within genomic "dark matter".

Programmed DNA elimination (PDE) is a notable exception to genome integrity, characterized by significant DNA loss during development. In many nematodes, PDE is initiated by DNA double-strand breaks (DSBs), which lead to chromosome fragmentation and subsequent DNA loss. However, the mechanism of nematode programmed DNA breakage remains largely unclear. Interestingly, in the human and pig parasitic nematode Ascaris, no conserved motif or sequence structures are present at chromosomal breakage regions (CBRs), suggesting the recognition of CBRs may be sequence-independent. Using Hi-C, we revealed that Ascaris CBRs engage in three-dimensional (3D) interactions before PDE, indicating that physical contacts between break regions may contribute to the PDE process. The 3D interactions are established in both Ascaris male and female germlines, demonstrating inherent genome organization associated with the CBRs and to-be-eliminated sequences. In contrast, in the unichromosomal horse parasite Parascaris univalens, transient pairwise interactions between neighboring CBRs that will form the ends of future somatic chromosomes were observed only during PDE. Intriguingly, we found that Ascaris PDE, which converts 24 germline chromosomes into 36 somatic ones, induces specific compartmentalization changes. Remarkably, Parascaris PDE generates the same set of 36 somatic chromosomes, and the 3D compartment changes following PDE are consistent between the two species. Overall, our findings suggest that CBRs spatially demarcate the retained and eliminated DNA and may contribute to their spatial organization during Ascaris PDE. We also demonstrated that the 3D genome reorganization of the somatic chromosomes in these nematodes following PDE is evolutionary and developmentally conserved.

Tick-borne diseases represent a significant threat to human and animal health worldwide. In the United States, the blacklegged tick, Ixodes scapularis (I. scapularis), serves as a competent vector for several bacterial pathogens, including Ehrlichia muris eauclairensis (EME). The I. scapularis embryonic cell line (ISE6) is a valuable tool for propagating tick-borne pathogens and studying tick-pathogen interactions. In this study, we examined the cellular complexity of ISE6 cells and their response to EME infection. Single-cell RNA sequencing revealed 15 distinct cell clusters present. Although ISE6 cells are heterogeneous, they do not display transcriptional similarity to any known tick tissues. Notably, this lack of similarity did not influence their susceptibility to EME infection. Our results demonstrated that EME infection induces time-dependent transcriptional changes in ISE6 cells: early infection is characterized by upregulation of genes associated with stress adaptation, mitochondrial function, and metabolic pathways, whereas late infection leads to broad downregulation of genes involved in the cell cycle, DNA replication, and cytoskeletal organization. These findings enhance our understanding of ehrlichial interactions with ISE6 cells and reinforce the utility of this cell line as a resource for isolating and propagating arthropod endosymbionts and tick-borne pathogens. IMPORTANCEThis study provides a single-cell resolution framework for interpreting tick cell line biology during infection with a medically relevant ehrlichial pathogen. Using scRNA-seq, we show that the I. scapularis embryonic-derived ISE6 cell line comprises multiple transcriptionally distinct cell states, yet these states do not map cleanly onto canonical tick tissue signatures, even when compared against a curated reference tissue atlas. Despite this heterogeneity, EME broadly infects ISE6 cell population, indicating that susceptibility is not restricted to a specific cell type. We further define a time-dependent arthropod vector response in which early infection is marked by activation of stress and metabolic adaptation response, followed by late-stage inhibition of key signaling, transcriptional, and proliferative pathways as bacterial burden increases. Together, these findings strengthen the biological interpretation of ISE6 as an in vitro model for tick-pathogen interactions and provide a resource for future mechanistic studies of ehrlichial persistence, replication, and vector competence.

The common pine sawfly, Diprion pini, is a widespread defoliator of pine forests across Europe and Asia, with outbreaks causing substantial ecological and economic damages. However, genomic resources for this species have been limited, hindering advances in molecular ecology or pest management. Here, we present a near chromosome-level reference genome for D.pini, generated using PacBio HiFi reads, Oxford Nanopore MionION long reads, and 10x Genomics linked reads. The final assembly is organized into mostly chromosome-sized scaffolds. It spans a length of 268 Mb, comprises 81 scaffolds, and has a scaffold N50 of 18.7 Mb. BUSCO analysis (hymenoptera_odb10) indicates a high genome completeness of 97.2%. With 22,7 kb the mitochondrial genome is unusually large due to an extended non-coding control region (6,874 bp). Gene prediction identified 26,335 protein-coding genes, of which 12,769 were functionally annotated. Comparative analyses with other sawflies and Apocrita identified 2,472 proteins unique to D. pini, some of which are putatively associated with the processing of plant secondary metabolites. Notably, our genome assembly highlights that, when a closely related, high-quality reference genome is available, chromosome-scale assemblies can be generated without the need of Hi-C sequencing. The genome provides a valuable foundation for the development of improved monitoring and management strategies for D. pini outbreaks and contributes to advancing fundamental research on Hymenoptera evolution.

Sporotrichosis, a subcutaneous mycosis caused by dimorphic fungi of the Sporothrix genus, has become a major zoonotic epidemic in South America, primarily driven by Sporothrix brasiliensis. To elucidate the genomic basis of its emergence and antifungal adaptation, we analyzed whole-genome sequences from 94 Sporothrix isolates, integrating single-nucleotide polymorphism (SNP), copy number variation (CNV), and genome-wide association (GWAS) analyses. Comparative genomics revealed 610,242 SNPs within S. brasiliensis and 1,474,627 within S. schenckii, confirming a marked disparity in intraspecific diversity. Phylogenomic tree inference resolved six well-supported S. brasiliensis clades with limited internal divergence, reflecting recent population expansion, while S. schenckii displayed deep phylogeographic structure separating North and South American lineages. CNV profiling identified 158 affected genes in S. brasiliensis (60 gains, 98 losses) and 88 in S. schenckii (54 gains, 34 losses), concentrated near sub-telomeric regions. In S. brasiliensis, gains were enriched for kinases and intracellular trafficking functions, whereas losses involved genes related to translation and primary metabolism, suggesting regulatory reinforcement coupled with metabolic streamlining. A GWAS of itraconazole resistance identified 81 SNPs distributed across multiple scaffolds, with many located within genes related with transport, signaling, and redox balance, supporting a polygenic basis for azole response. Together, these results highlight distinct evolutionary strategies of closely related Sporothrix species and delineate the genomic changes associated with the emergence and drug tolerance of S. brasiliensis.

In Toxoplasma gondii, microneme proteins (MICs) are secreted components of the apical complex that play central roles in motility, host cell attachment, and invasion. Because proteins at the host-parasite interface are often predicted to evolve rapidly, MICs have been suggested as candidates for adaptive diversification. We tested this expectation using comparative analyses of three relatively understudied microneme proteins, MIC13, MIC12, and MIC16. Coding sequences were assembled from GenBank and ToxoDB, aligned by translation, and analyzed using maximum-likelihood phylogenetics, codon-based tests of selection, and predicted protein structure. MIC13 was represented by 51 sequences, MIC12 by 30, and MIC16 by 34, spanning multiple T. gondii haplogroups and including Hammondia hammondi and Neospora caninum as outgroups. All three genes were highly conserved among T. gondii strains, but their phylogenetic trees were topologically incongruent, indicating that individual MICs do not recover a single shared strain history. Contrary to expectation, no positively selected codons were detected in any gene. Instead, purifying selection was detected at one site in MIC13 and 15 sites in MIC12, while no significant codon-specific selection was detected in MIC16. Several constrained MIC12 sites overlapped annotated EGF and calcium-binding EGF-like domains, consistent with structural conservation of extracellular adhesion modules. AlphaFold prediction of MIC13 supported two sialic acid-binding micronemal adhesive repeat regions, but the single constrained MIC13 site did not overlap these motifs. Together, these results indicate that MIC13, MIC12, and MIC16 are shaped more by sequence conservation and heterogeneous gene histories than by strong recurrent positive selection. These findings refine expectations for microneme evolution in T. gondii and highlight conserved domains that may be important for parasite invasion and future functional study.

Quantifying feeding behavior with high temporal and spatial precision is critical for understanding how internal state, sensory cues, and neural activity shape food intake and dietary choice. Here, we describe a detailed protocol for performing consumption and dietary choice assays in Drosophila using the flyPAD/optoPAD system. This method enables simultaneous measurement of feeding events across multiple arenas while allowing precise control of gustatory stimuli and optogenetic stimulation. We provide step-by-step instructions for assay food preparation, flyPAD arena setup, data acquisition, and downstream data organization with suggested analyses. This approach is suitable for studying consumption, nutrient preference, learning, and state-dependent modulation of feeding behaviors, and can be readily adapted for optogenetic manipulations and comparative choice assays.