Orientia tsutsugamushi (Ot), the causative agent of scrub typhus, is an obligate intracellular bacterium naturally maintained in Leptotrombidium mites, yet its interactions within arthropod hosts remain poorly understood. Here, we employed two tick cell lines, Ixodes scapularis ISE6 and Rhipicephalus microplus BME/CTVM23, as arthropod models to investigate the intracellular lifecycle of Ot strains TA686 and Karp. Both strains efficiently infected tick cells and replicated within them, with ISE6 supporting more robust growth. Electron microscopy images revealed that Ot maintains its characteristic cytoplasmic, non-vacuolar location in tick cells and exits infected cells by budding off the surface in a membrane-encased structure. Time-course immunofluorescence imaging demonstrated progressive intracellular replication and dynamic expression of Ot outer membrane autotransporters ScaA and ScaC, with ScaC enriched early in infection and ScaA at later stages. A microscopy-based analysis of metabolic activity using a clickable methionine analog L-homopropargylglycine showed that high ScaA abundance correlated with reduced translational activity, suggesting a link between ScaA abundance and late-stage or extracellular-like developmental states. The subcellular location of Ot in tick cells differs from the characteristic dynein-driven perinuclear clustering observed in mammalian cells and was not sensitive to disruption of microtubules, suggesting a distinct mode of distribution. Together, these findings identify tick cell lines as tractable and biologically relevant arthropod models for studying Ot and dissecting host-microbe interactions.IMPORTANCEOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium naturally maintained in trombiculid mites, yet its interactions with arthropod host cells remain poorly understood. This study establishes, for the first time, a tractable in vitro arthropod model for O. tsutsugamushi using tick cell lines from Ixodes scapularis and Rhipicephalus microplus. We demonstrate that both virulent and avirulent Orientia strains efficiently infect and replicate in these cells while maintaining their characteristic cytoplasmic location and budding mode of exit. Distinct temporal expression of the bacterial autotransporters ScaA and ScaC and their relationship to metabolic activity reveal stage-specific adaptations during infection. These findings provide essential mechanistic insight into how O. tsutsugamushi persists and develops within arthropod hosts. The tick cell model enables comparative studies of host-pathogen interactions across vector species and offers a powerful tool to investigate transmission biology and potential targets for intervention.
Pinyon-Juniper (PJ) woodlands are critical arid habitats that host diverse biotic communities. We examined tree-arthropod relationships across elevational gradients in northern Arizona, comparing foliar and trunk communities on the 2 dominant tree species, pinyon and juniper. Understanding these dynamics is vital for climate-change adaptation and biodiversity management. Using trunk-refugia traps and sweep-netting, we sampled arthropods on trunks and foliage of both hosts and tested how host tree, elevation, and precipitation structured assemblages. We hypothesized (i) trunk and foliar communities would be compositionally distinct; (ii) communities would differ between pinyon and juniper because of host-specific taxa rather than dominance by a single tree species; and (iii) peaks in foliar diversity would occur where cooler, wetter conditions coincided (mid-elevation sites). The study confirmed distinct foliar and trunk communities and strong, taxon-specific host-tree effects. Juniper foliage supported higher foliar richness and diversity in both years, whereas pinyon trunks supported higher arthropod abundance and several trunk-associated taxa. Pinyons and junipers therefore supported unique trunk and foliar assemblages, contradicting previous studies on ground arthropods. We observed host-specific preferences, with spiders, leafhoppers, and parasitoid wasps more common on juniper foliage, while mites were more abundant in pinyon foliage. Climate associations were year- and taxon-specific: precipitation and elevation structured foliar richness in 2019 but were weaker in 2020. Our findings show that host-tree identity was the more consistent predictor in this dataset, whereas elevation and precipitation proxies captured site-level associations with long-term climatic context rather than year-specific weather, providing a spatial baseline for future monitoring under vegetation and climate change.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays a crucial role in the regulation of detoxification enzymes contributing to insecticide resistance in insects, whereas its function in growth and development remains poorly understood. This study systematically investigated the biological functions of the BmAHR gene in the lepidopteran model insect, Bombyx mori. The BmAHR gene was identified through cloning and characterized. Temporal and spatial expression profile analysis revealed that BmAHR was highly expressed in the adult wings and antennae. BmAHR mutants were obtained by a binary transgenic CRISPR/Cas9 system. The study revealed that BmAHR deficiency resulted in severe wing developmental abnormalities in mutant adults. Moreover, the absence of BmAHR disrupted the normal development of the antennae, which further impaired mating behavior in adults. RNA-seq and qRT-PCR analyses uncovered that mutation of BmAHR simultaneously disrupted signaling pathways required for antennal development and altered the transcriptional regulation of olfactory receptor genes. Our findings provide novel insights into the biological function of BmAHR in antennal development, highlighting its essential role in shaping antennal structure and modulating olfactory gene expression, thereby influencing mating behavior. Collectively, our study underscores the AHR gene as a potential molecular target for pest control.
The development of insect appendages is governed by deeply conserved genetic programs, even as developmental strategies diverge widely across taxa. In this study, we identify the conserved transcription factor Grainyhead (Grh) as a crucial regulator of insect wing development. In the silkworm Bombyx mori, CRISPR/Cas9-mediated knockout of BmGrh did not compromise larval viability but resulted in severe wing defects in adults, including crumpled and non-expandable wings. Similarly, tissue-specific knockdown of DmGrh in Drosophila melanogaster wing imaginal discs led to pupal lethality, a sex-biased eclosion rate, and adults exhibiting crumpled wings with disrupted wing hair polarity. Comparative transcriptomics revealed that loss of DmGrh function predominantly downregulates genes associated with cuticle structure and extracellular matrix organization in both species. In Drosophila, chromatin immunoprecipitation further confirmed that DmGrh directly binds to regulatory regions of these downregulated cuticle-related genes. Through functional screening, we identified the cuticle protein gene cpr65Ea as a key downstream effector, whose knockdown recapitulated the wing morphogenesis and eclosion defects observed in DmGrh RNA interference individuals. Moreover, extending our investigation to an agricultural pest, RNAi-mediated silencing of SfGrh in the white-backed planthopper (Sogatella furcifera) impaired wing expansion and completely abolished flight ability. Our findings establish Grh as an evolutionarily conserved regulator of insect wing development and highlight its potential as a novel genetic target for pest management by disrupting flight capacity and dispersal.
Synapses are specialized structures for information exchange between neurons and their targets, and precise regulation of synaptic growth is crucial for the formation and plasticity of neural circuits. The neuromuscular junction (NMJ) of fruit fly larvae is an excellent model for studying molecular mechanisms underlying synaptic development. There are few reports on the role of long noncoding RNAs (lncRNAs) in synaptic development at NMJ. Here, we reported a lncRNA, Synapse Development Regulatory Gene (SDRG), which regulates synaptic growth by antagonizing frequenin 2 (frq2) through Coracle (Cora). SDRG deficiency induced synaptic overgrowth characterized by excess satellite boutons at NMJ terminals, and simultaneously high frequenin 1 (frq1) and frq2 RNA levels. Genetically, frq2, not frq1, knock-down driven by motoneuron-specific Gal4 could rescue this growth defect, which was mediated by Cora protein. At the molecular level, SDRG promotes the recruitment of Cora protein to frq2 RNA. Our work exhibits a new function of lncRNA and is beneficial for unveiling the pathogenesis of neuropsychiatric disorders with abnormal synaptic development.
Superposition compound eyes are a hallmark of many nocturnal beetles and moths and represent a major evolutionary innovation for dim-light. Although their optical organization has been extensively documented, the developmental processes underlying their assembly remain poorly understood. Here, we traced the morphogenesis of the compound eyes in the diamondback moth Plutella xylostella from the prepupal stage to adult emergence using transmission electron microscopy. Early ommatidia adopt an apposition-like arrangement, with crystalline cones in direct contact with distal retinulae. The retinulae subsequently undergo coordinated proximal displacement, while secondary pigment cells expand into the intervening space, forming a pigment-free clear zone. The tracheal tapetum develops via distal extension of numerous tracheoles between adjacent retinulae, developing in close synchrony with the formation of the clear zone. The rhabdom elongation proceeds in a distinct distal-to-proximal sequence, and corneal nipples appear prior to deposition of fibrous corneal lamellae. These observations demonstrate that the superposition eyes of P. xylostella arise through stepwise modification of an apposition-like developmental framework rather than through the formation of entirely novel structures, supporting the view that evolutionary innovation in insect compound eyes primarily results from modification of conserved developmental programs.
Crustacean and molluscan processing as seafood generates a significant amount of shell waste annually, owing to the dense, mineral-rich composition of the exoskeleton. Although valorization efforts have mostly focused on chitin and chitosan, the protein fraction remains underutilized despite its prominent nutritional, functional, and sensory potential. Crustacean shells typically contain 30%-40% protein, enriched with bioactive and flavor-active amino acids, such as glycine, proline, alanine, and arginine. These proteins exhibit promising functional properties, including solubility, emulsification, foaming capacity, and stability. However, efficient recovery remains challenging due to the tightly bound chitin-protein-CaCO3 matrix and structural complexity of the Bouligand-type structure of crab exoskeleton. However, mollusc shells are primarily mineralized structures composed of calcium carbonate (95%-99%), with a smaller organic fraction (1%-5%) consisting mainly of proteins, glycoproteins, and polysaccharides. Compared with crustacean shells, molluscan shells have lower protein content. This review critically evaluates the shell protein valorization and extensive data on composition and extraction strategies, such as green methods. The multifunctionality of shell-derived compounds also extends to pharmaceutical, biomedical, and environmental domains. Although research on shell valorization is expanding, critical gaps remain in techno-economic feasibility, allergenicity assessment, regulatory clarity, and the development of scalable processing techniques. Overcoming these barriers requires integrated efforts across food science, biotechnology, and sustainability practices. By integrating green extraction technologies, compositional profiling, and food system applications, this review advances innovative and sustainable pathways for crab shell valorization aligned with circular bioeconomy principles.
The transcription factor T-box20, a member of the conserved T-box gene family, plays crucial roles in the growth and development of various organisms. In this study, we identified a T-box family member, T-box20-like (Tbx20b) in Spodoptera frugiperda. Evolutionary analysis revealed that Tbx20b is a duplicated paralog generated via gene duplication events and identified a unique G-quadruplex (G4) structure in the upstream promoter region. Biochemical and cellular assays confirmed that G4 formation in the Tbx20b promoter regulates its transcriptional expression. CRISPR/Cas9-mediated knockout of either the Tbx20b gene or its promoter G4 motif resulted in significant downregulation of Tbx20b expression, accompanied by impaired growth, defective metamorphosis, and reduced reproductive capacity in S. frugiperda. Comparative transcriptomics suggests that the Tbx20b may function in fatty acid metabolism. Collectively, these findings uncover a G4-dependent transcriptional regulatory mechanism governing Tbx20b expression and provide novel insights into the functional roles of promoter G4 structures in regulating key developmental processes in lepidopteran insects.
This study describes the organization of the antiparallel sperm bundle and the ultrastructure of the spermatozoa of the beetle Zophobas confusa Gebien, 1906. The first peculiar characteristic is observed in spermiogenesis through the orientation of the spermatid nuclei, which align in opposite directions within the testicular cysts at the beginning of development. The mature, filiform spermatozoon of Z. confusa measures approximately 107 µm in total length, with an elongated, tapered nucleus measuring about 18 µm. The sperm head is composed of a bilayered acrosome and a nucleus featuring a distinctive crescent-shaped invagination at its posterior base. The flagellum is characterized by the conserved axoneme of 9 + 9 + 2 microtubules, flanked by two large, asymmetric mitochondrial derivatives containing prominent paracrystalline inclusions, and two symmetric, elongated accessory bodies. The structural complexity of the flagellum simplifies towards the distal end. Cross-sections of testicular cysts reveal axonemes with both clockwise and counterclockwise orientations, a direct result of the antiparallel arrangement. We highlight that such an arrangement might be established even before spermiogenesis, as early as the second meiotic division of spermatocytes, based on observed sperm abnormalities. The combination of conserved tenebrionid features, such as symmetrical accessory bodies, with unique traits, such as nuclear base invagination, solidifies the phylogenetic position of Z. confusa within the Tenebrionidae. Together with the displayed bipolar sperm arrangement, it reinforces this as a key synapomorphy for derived Tenebrionoidea, distinguishing them from basal lineages.
Edible insects have emerged as a sustainable alternative protein source, yet their widespread application in food systems is constrained by well-established allergenic risks. Accumulating evidence indicates that insect proteins cannot be assumed to be inherently low-allergenic, largely due to the presence of highly conserved pan-allergens sharing significant sequence homology and extensive cross-reactivity with crustaceans and mites. This review provides a comprehensive and mechanism-oriented overview of the physicochemical characteristics, immunological determinants, and processing-induced structural alterations of major insect allergens, with a particular focus on tropomyosin, arginine kinase, and other clinically relevant allergenic proteins. We critically examine how protein stability, epitope distribution, and digestion resistance govern IgE-binding capacity and cross-reactive immune responses. Current allergenicity mitigation strategies, including enzymatic hydrolysis, thermal processing, non-thermal physical technologies, and chemical or structural modifications, are systematically discussed with respect to their efficacy, limitations, and impacts on nutritional and sensory quality. Particular emphasis is placed on emerging synergistic approaches that integrate structural destabilization with targeted enzymolysis to achieve more effective allergenicity reduction. Finally, future perspectives are proposed, highlighting the need for precision regulation of allergenic proteins at the epitope and structural-domain levels, standardized allergenicity assessment frameworks, and improved translational relevance between in vitro assays and clinical outcomes. By integrating molecular mechanisms with food processing strategies, this review aims to provide a scientific basis for the development of safer, nutritionally adequate, and consumer-acceptable insect-based foods.
Expansion microscopy (ExM) enlarges biological samples by embedding them in a swellable hydrogel, enabling nanoscale imaging of subcellular structures with standard light microscopes. This offers an accessible alternative to super-resolution methods. ExM has yet to be combined with the Oligopaint DNA fluorescence in situ hybridization (FISH) technology. We present an optimized ExM workflow for simultaneous Oligopaint DNA FISH and immunofluorescence (IF) in intact Drosophila ovaries. The protocol incorporates nucleic-acid anchoring, reliable protein retention, and digestion conditions that preserve chromatin while maintaining probe accessibility. Our approach achieves ~ 5 × expansion and strong signal retention, enabling high-resolution studies of nuclear organization.
Morphogenesis requires dynamic changes in cell and tissue mechanics. Because organs are complex multi-cellular structures that develop slowly, understanding how they acquire their unique 3-D forms requires in vivo experimental systems that can bridge the discrete processes that occur at different physical-scales, from cell, to tissue, to multi-organ, and at different time-scales, from minutes, to hours, to days. The challenge of establishing causal relationships across scales has limited exploration of the cell biological processes that collectively produce the changes in tissue material properties and mechanics required for developing epithelia to deform in stereotyped ways. In this review, we discuss recent work focused on the Drosophila pupal retina that has begun to fill this gap. The new findings collectively emphasize how supracellular networks can channel mechanical outputs generated from rapid, cytoskeletal/junctional dynamics into the organized changes in tissue-scale material properties and mechanics needed to produce functional 3-D organ morphologies. By linking cellular dynamics with tissue-scale mechanics, supracellular networks may also critically determine how tissue-extrinsic forces from the growth environment contribute to programmed shape deformations that occur during morphogenesis. The mechanisms that emerge from this collective body of work provide a valuable conceptual framework for considering how cell and tissue mechanics, working in combination with environmental forces and tissue-intrinsic force generation programs, may instruct and constrain 3-D morphologies across a broad range of biological contexts.
Litopenaeus vannamei, the most widely farmed crustacean, relies on family-based selection where accurate pedigree information is essential. Although SNP-based tools offer high-accuracy pedigree assignment, adoption in commercial breeding remains limited. In this study, we developed a commercially viable 1K SNP panel with 1125 markers. Markers were selected from a 55K SNP dataset comprising 2330 individuals. We established a practical pedigree reconstruction workflow and implemented the panel in a field breeding population. The population included a selection group where families were reared separately and a test group where individuals were communally reared. We introduced anchor individuals from the selection group to enable pedigree linkage. All 1818 individuals from 72 families were accurately assigned. Family reconstruction achieved 100% consistency with known records, even when parents were partially missing. Heritability estimates for harvest weight ranged from 0.32 to 0.36 using pedigree-based BLUP (PBLUP), genomic BLUP (GBLUP), and single-step genomic BLUP (ssGBLUP). The ssGBLUP model, using a 0.15 to 0.85 weighting of G and A, achieved 6.67% and 19.40% higher accuracy than PBLUP and GBLUP. The panel also supported population structure analysis and diversity monitoring, demonstrating its value for genomic evaluation in commercial L. vannamei breeding.
The ryanodine receptor (RYR) genes encode evolutionarily conserved calcium release channels involved in a wide range of calcium-dependent biological processes. Here, we show that the sole Drosophila RYR gene (dRyR) functions in differentiated somatic and cardiac muscle as well as in developing embryonic myotubes. In the larval body wall muscles, dRyR protein localizes at the SR membranes, and dRyR knockdown adversely affects muscle contractility, suggesting its conserved role in calcium-triggered E-C coupling. After dRyR attenuation, sarcomere, and mitochondrial patterns are severely impaired, showing dRyR involvement in structural muscle properties. However, dRyR is also prominently expressed and functionally required in growing embryonic muscles. dRyR loss of function leads to myotube growth defects and thin myofiber phenotypes, while its overexpression induces myofiber splitting. Given the structural and functional conservation of dRyR, we used Drosophila to test the impact of one human RYR1 variant of unknown significance (VUS). Larvae carrying p.Met4881Ile RYR1 VUS showed impaired mobility and altered structural muscle properties reminiscent of those seen in dRyR knockdown, thus indicating it is likely pathogenic. Overall, we show that Drosophila dRyR plays a conserved role in setting muscle contractility and structural muscle features. Our findings underline the still under-investigated role of dRyR as a promyogenic factor and provide a first example of the impact assessment of a human RYR1 VUS in Drosophila.
Vegetated field borders are widely promoted as tools to enhance biodiversity and strengthen biological control in agroecosystems. However, their role in pest dynamics remains conceptually fragmented and empirically inconsistent. Here, we develop a unified framework explaining how crop border vegetation influences pest populations through four interlinked ecological mechanisms. First, borders act as host reservoirs and selective filters, providing alternative hosts and overwintering habitat that enhance pest persistence across crop cycles. Second, borders modify pest colonization dynamics by shaping movement, aggregation, and host-location behavior at crop edges. Third, borders restructure multitrophic networks, simultaneously supporting natural enemies, alternative prey, vectors, and pathogens, generating nonlinear effects on pest suppression. Fourth, repeated disturbance and management function as selective filters, determining which plant functional groups dominate borders and, consequently, which pest and natural enemy communities are maintained. To ground this framework, we conduct a structured synthesis of published empirical and conceptual studies on crop-border vegetation, including weed and arthropod surveys, and classify them according to the proposed mechanisms. Our synthesis reveals a strong emphasis on multitrophic effects, whereas colonization processes and disturbance filtering are comparatively underexplored. Across mechanisms, plant identity and dominance structure consistently emerge as stronger predictors of pest outcomes than species richness alone. We argue that borders are not inherently beneficial or harmful but function as selectively structured ecological interfaces shaped by management history and species composition. By integrating temporal persistence, spatial behavior, network interactions, and anthropogenic filtering, our framework provides a predictive basis for IPM-oriented design of field borders, enabling management strategies that reduce pest carryover, disrupt colonization pathways, and enhance biological control while maintaining ecosystem services. This article is part of the theme issue "The Biology, Ecology, and Management of Plant Pests".
Parasitism is a key ecological phenomenon that can strongly influence population dynamics and community structure in marine ecosystems. This study assessed the prevalence of the rhizocephalan parasite Peltogasterella gracilis in the hermit crab Pagurus edwardsii across five coastal localities in the Biobío Region, Chile, and evaluated its effects on host morphometry. A total of 134 crabs were collected, of which 30 (22.2%) were parasitized, showing significant geographic variation, with Lirquén and Cocholgüe exhibiting the highest infection rates. No significant differences in parasitism prevalence were observed between males and females; however, none of the parasitized females were ovigerous, supporting the hypothesis that Pe. gracilis inhibits female reproductive activity. Morphometric analyses revealed that parasitized individuals had proportionally wider cephalothoraxes than non-parasitized crabs of equivalent length, with no evidence of sexual dimorphism in this relationship. Additionally, a moderate positive correlation was observed between the number of externae and relative host carapace length, reflecting the influence of parasite development on host morphology. Externae exhibited considerable variability across individuals and localities, including differences in size, shape, and coloration, likely reflecting phenotypic variability driven by a combination of host condition, parasite developmental stage, and local environmental factors. Overall, our results indicate that, Pe. gracilis occurs across multiple localities within the studied region, suggesting a spatially structured distribution rather than restriction to a single site. In addition, Pe. gracilis exerts significant effects on the morphometry and reproductive capacity of Pa. edwardsii, with potential implications for host population dynamics. These findings underscore the need for further research to elucidate the mechanisms underlying parasite-induced functional castration and to deepen understanding of this host-parasite system.
Haemaphysalis longicornis poses severe global veterinary and public health threats. Its obligate nutritional symbiont, Coxiella R1 (CLE), critically influences reproduction and development. Understanding the interplay between tick genetic diversity and CLE abundance is essential for developing symbiont-targeted control strategies. Haemaphysalis longicornis ticks were collected from 11 locations across nine Chinese provinces. Population genetic diversity and structure were analyzed based on simple sequence repeat (SSR). Coxiella R1 relative abundance in individual ticks was quantified via qPCR. Extensive genetic diversity was detected across Chinese H. longicornis populations using three validated polymorphic SSR markers: mean Ad = 5.667, GD = 0.7052, and PIC = 0.6646. Population structure analysis (K = 2) revealed two distinct genetic clusters. Ticks from Shanghai (Chongming Island) and Jiangsu (Xuzhou City) formed a genetically distinct group, significantly separated in PCoA from populations in Liaoning, Sichuan, Hubei, Shaanxi, Anhui, Jiangxi, and Zhejiang. Phylogenetic analysis supported this clustering but indicated limited geographic structuring overall. Crucially, Coxiella R1 was ubiquitous in all populations, but its abundance varied significantly between regions (P < 0.05, Kruskal-Wallis/Dunn's). CLE levels were highest in Liaoning and Shanghai ticks and significantly lower in those from Jiangsu and Zhejiang provinces. Despite the shared genetic ancestry of Shanghai and Jiangsu ticks, their CLE burdens were markedly different. This study demonstrates substantial genetic diversity within Chinese H. longicornis populations and defines a distinct genetic cluster including ticks from Jiangsu and Shanghai. This variation in CLE burden showed no association with the identified tick population genetic structure or geographic distance.
The Drosophila gene uninflatable (uif) encodes a conserved insect protein, first identified for its roles in the development and endopolyploid growth of several larval tissues. Uif is a transmembrane protein and its large extracellular domain contains several protein-protein interaction motifs, including multiple EGF (Epidermal Growth Factor-like) repeats. More recent studies have established that Uif can interact with Notch, a major regulator of Drosophila growth and differentiation, through its EGF repeats and have identified ways that Uif binding can modify Notch behavior. Endocytosis of Notch-Uif complexes into a particular class of endosomes has also been identified and implicated in cell fate decisions in the sense organs of the notum (thorax). We have examined uif's functions in the Drosophila wing to assess possible roles in 1) growth of a mitotically derived tissue, 2) cell fate decisions in specialized wing structures, and 3) Notch-dependent processes. We used previously characterized Gal4 lines and RNAi constructs to suppress uif and Notch in different wing compartments. In addition to a role in mitotic growth throughout the wing, we have identified two new uif activities that are also shared with Notch: 1) regulation of pigment synthesis within the wing cuticle and 2) control of chemosensory sense organ number in the anterior wing margin through a role in an apoptosis-related mechanism. However, uif does not participate in two roles of Notch that regulate cell fate decisions: sense organ differentiation and formation of wing vein tissue. Given the similarities in the development of the notal and wing margin sense organs, we investigated further the previously proposed role for uif in differentiation of these structures on the notum. We found that loss of uif affects the growth of the bristles of the notal microchaete sense organs, but not their differentiation.
With accelerating biodiversity loss, tracking both inter- and intraspecific diversity is critical, as within-species variation underpins population viability and adaptive potential. We developed a new multi-locus framework, and obtained an Orthoptera-specific marker panel targeting 398 nuclear loci plus the mitogenome and ribosomal DNA to assess species boundaries, population structure and intraspecific diversity. We applied this approach to 645 specimens covering all 105 Swiss Orthoptera species, sampled during the nationwide Red List update. This multi-locus dataset produced a well-supported phylogeny and resolved several taxonomic ambiguities with direct conservation relevance. We demonstrate its ability to identify fine-scale population structure and further illustrate its application to genetic diversity estimation. While species' IUCN threat levels were not correlated with genetic diversity, we found a significant negative association between genetic diversity and dependence on riparian habitats: river-bank specialists showed lower diversity, likely reflecting the severe fragmentation and alteration of these ecosystems. Our results demonstrate that the application of new molecular tools provides valuable, complementary insights not only for taxonomy but also for conservation assessments. The development and application of genetic monitoring frameworks, such as the one presented here, are highly valuable for promoting the inclusion of genetic diversity in future conservation assessments.
Brain tumor (Brat) is a Drosophila TRIM-NHL protein required for embryogenesis and neural stem cell differentiation. Although structural and biochemical studies established that the Brat NHL domain specifically binds RNA, the in vivo requirement for this activity has not been directly tested. Here, we used structure-guided mutagenesis and genome engineering to determine whether RNA recognition is essential for Brat function during development. The direct interaction between Brat's NHL domain and RNA containing Brat Binding Sites (BBS) can be abolished by alanine substitution of three separate residues on the NHL surface. We introduced these point mutations into the endogenous brat locus by CRISPR-mediated Scarless Gene Editing to generate three independent RNA-binding defective mutant (RBDmt) alleles. Complementation tests demonstrated that each allele behaves as a strong loss-of-function mutation: homozygotes and hemizygotes are inviable, and RBDmt alleles fail to complement classical brat null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known brat alleles. Consistent with the namesake brat phenotype, RBDmt larval brains exhibited widespread expression of neuroblast markers and a marked reduction of neuronal differentiation. In embryos, these alleles failed to complement female sterile brat alleles and recapitulated characteristic abdominal segmentation defects. Finally, RT-qPCR showed increased expression of endogenous Brat target mRNAs in mutant larvae, consistent with loss of Brat-mediated repression. Together, these results demonstrate that direct RNA binding is an essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.