BMC Biology

Isopods are an order of crustaceans characterised by a biphasic moulting pattern, in which the posterior cuticle is shed before the anterior cuticle, with an intramoult period of up to a few days between the two. In order to understand how this unusual moulting pattern is regulated, we carried out a transcriptomic analysis covering three distantly related terrestrial isopod species. We analysed the transcriptomic profile of four body regions: the front legs, the hind legs, the thorax and the head, at different phases of the moulting cycle in the three species. We describe a conserved cyclic pattern in the transcriptomic profiles corresponding to the phases of the moulting cycle. The genes driving this conserved pattern provide a catalogue of the central players of the moulting process and are prime candidates for future experimental work. Furthermore, we show that during the intramoult phase, the posterior limbs display a transcriptomic profile more similar to the postmoult phase, indicating that at this phase, the animal is functionally a transcriptomic chimera, with the anterior and posterior halves experiencing radically different molecular environments, with disjunct regulatory programmes active in each half.

Exposure to the potassium channel blocker barium chloride (BaCl2) causes head degeneration in Dugesia japonica flatworms, followed by regeneration of BaCl2-insensitive heads, offering a unique model for studying transcriptional resilience to novel stress. We performed RNA sequencing on individual planaria to investigate different transcriptional solutions to the BaCl2 challenge, and how regeneration history and social environment shape transcriptomic responses to BaCl2. We identified a robust transcriptional strategy and a potential sub-strategy for enabling BaCl2-insensitive head formation. Moreover, we observed pronounced transcriptional differences between untreated worms regenerating from tail fission fragments (tail-regenerated), and untreated full-sized worms that did not fission during the experiment (intact controls), highlighting the lasting impact of regeneration history. Relative to controls, tail-regenerated worms upregulated neurodevelopmental and morphogenetic programs, while downregulating mitochondrial transport and stress-response pathways. Relative to intact controls, BaCl2-exposed regenerates upregulated ion transport, metabolic, cell cycle, and inflammatory pathways, while downregulating neuronal signaling, ion homeostasis, morphogenesis, and tissue repair programs. Comparison of BaCl2-exposed isolated and BaCl2-exposed group-housed worms revealed minimal transcriptional divergence between social conditions. These findings underscore the complex interplay between regeneration, chemical stress, and social context in shaping gene expression.

Where recombination takes place is crucial as it determines the position of genetic reshuffling, which facilitates species evolution and adaptation. In many vertebrates, PRDM9 determines the location of double-strand breaks that initiates meiotic recombination. However, this only applies for Prdm9 orthologs that possess certain molecular features, such as the presence of the four functional domains (KRAB, SSXRD, PR/SET and ZnF), the conservation of catalytic tyrosines and a fast-evolving zinc finger array. Although the evolutionary origin of PRDM9 has been inferred to the last common ancestor of metazoans, it has been poorly explored outside vertebrates. Here, we explored the genomes of 28 cnidarian species and identified, for all of them, at least one full-length ortholog that appeared to be functional. Striklingly, ten out of the 28 species carried several full-length paralogs with the same molecular markers of functionality. This pattern has never been observed in any other explored taxa. We also identified many truncated paralogs that arose at different times, thereby revealing an important birth-and-death dynamics of Prdm9 in cnidarians. Phylogenetic analysis revealed that the multiple full-length paralogs are all the result of recent duplication events, occuring mainly after speciation, although truncated paralogs arose all along cnidarian evolution. One of the oldest truncated paralogs appeared before the divergence between Actinaria and Scleractinia. This indicates the probable acquisition of a new function that has been conserved for at least 540 million years and whose nature is still unknown. In addition to providing insights into the ancient origins of PRDM9's function, our work now raises new questions about the functional redundancy of these multiple full-length paralogs, as well as their evolutionary significance for cnidarian genomes.

Regenerative capacity varies widely across flatworms (Platyhelminthes). Whereas catenulids, microstomids and planarians can regenerate a complete head de novo, other flatworms cannot. This striking diversity raises a longstanding evolutionary question: does whole-body regeneration represent an ancestral trait that was subsequently lost in multiple lineages, or did it evolve convergently? Addressing this question requires comparative analyses of the molecular mechanisms underlying regeneration across phylogenetically diverse flatworms. Here, we focus on Wnt signaling, a deeply conserved regulator of antero-posterior (A-P) patterning and a central determinant of head-versus-tail identity during planarian regeneration, to establish a mechanistic framework for such comparisons. Although Wnt signaling has been studied extensively in planarians and parasitic neodermatans, its evolution and deployment in other flatworm clades remain poorly characterized. To address this gap, we characterized the complement of Wnt signaling components in two early-diverging flatworm clades, Catenulida and Macrostomorpha, with particular emphasis on expression and function in the catenulid Stenostomum brevipharyngium. Phylogenetic analyses reveal the ancient loss of six Wnt families and one secreted Frizzled-related protein (sFRP) family in the last common ancestor of flatworms, followed by additional lineage-specific gene losses and expansions. Moreover, several Wnt pathway components display markedly divergent expression patterns between catenulids and other flatworms, while functional analyses indicate corresponding differences in their regenerative deployment. Together, our findings reveal a dynamic evolutionary history of the flatworm Wnt signaling toolkit and establish a comparative framework for testing whether the molecular circuitry underlying head regeneration is ancestrally conserved or has evolved independently in distinct flatworm lineages.

The Arabian killifish (Aphaniops dispar) is an excellent model for studying human diseases such as fungal infections and cancer progression. The embryos possess a transparent chorion for live imaging and display an extended period of independent feeding (13 days post-fertilisation). They also exhibit broad thermal tolerance which enables live imaging at physiologically relevant human temperatures. However, omics resources for this species remain limited and restricts its use in genomics studies. We generated a 1.45 GB high-quality reference assembly with 24 chromosome-level scaffolds and N50 score of 60.64 Mb by combining long-read, long-range, and short-read sequencing. The A. dispar genome exhibits a high level of homozygosity (95.6%) with BUSCO gene completeness scores of 99.2% and 97.5% for the Actinopterygii and Cyprinodontiformes lineages, respectively. The genome is highly repetitive (58.7%) and the majority of these are DNA transposons. The mitochondrial genome is 81.55% similar to that of Aphanius iberus (the Spanish toothcarp), with minor structural modifications. A. dispar exhibits highly conserved synteny with Fundulus heteroclitus within the Cyprinodontoidei clade. Together, these findings provide a valuable genomic resource for comparative genomics within the teleost community.

Resolving deep phylogenetic relationships requires integrating multiple lines of evidence, as distinct evolutionary forces shape signals from different genomic markers. Here, we investigate the systematics of the controversial APPD lineage (Anomiidae, Placunidae, Plicatulidae, and, by inference, Dimyidae) within Pectinida sensu lato using phylogenomic, comparative genomic, transcriptomic, proteomic, and morphological approaches. Our analyses consistently recover APPD as a monophyletic lineage sister to Limida and Pectinoidea, divergent at [~]428 Mya. With three novel high-quality genomes, extensive progressive chromosomal fusions demonstrate a reduction in chromosome number of the APPD lineage (6-13), compared with an ancestral 20 molluscan linkage groups (MLGs). Accompanied by extensive intrachromosomal gene-order scrambling, we identify one functional centromere in Placuna vitream flanked by two vestigial centromeric remnants on a single chromosome, providing a potential resource for investigating centromere inactivation and neocentromere formation. Mitochondrial genomes of APPD lineage exhibit unprecedented plasticity in translational decoding: Pododesmus employs the invertebrate mitochondrial code; Heteranomia employs +1 translational frameshifting to bypass in-frame TAG codons, whereas in Anomia, Enigmonia, Placuna, and Plicatulidae, TAA is reassigned to tyrosine and confirmed by proteomic evidence, which supports mitochondrial frameshifting in APPD lineage and defines a novel translation table for bivalves. Integrating phylogenetic distinctiveness, deep divergence, extreme karyotypic restructuring, unique mitochondrial features, and morphological diagnosability, we elevate the APPD lineage into Anomiida ord. nov. This revision resolves long-standing uncertainties for Pectinida sensu stricto and Limida, and establishes the APPD lineage as a valuable system for investigating chromosome fusion, centromere evolution, codon reassignment, and translational recoding. ClassificationBiological Sciences; Evolution SIGNIFICANCE STATEMENTWe have re-examined a controversial group of marine bivalves (Anomiidae, Placunidae, Plicatulidae, and Dimyidae). Our integrative approach shows that these animals split from scallops and their relatives more than 428 million years ago and have undergone drastic chromosomal fusions that reduced their chromosome number from 20 to as few as 6. Additionally, some species evolved unusual ways of reading their mitochondrial genetic code, either reassigning the stop codon to tyrosine or using +1 translational frameshifting to skip stop signals. The combination of deep evolutionary time and genomic divergence warrants recognizing them as a new order, Anomiida ord. nov. This work, as a case study, demonstrates how chromosome fusion and genetic code variation contribute to invertebrate diversity.

Large multigene families are a hallmark of trypanosomatid genomes and play major roles in genome plasticity and parasite adaptation. Among them, the Dispersed Gene Family 1 (DGF-1) of Trypanosoma cruzi remains one of the least understood because of its large size, repetitiveness, and fragmented annotations. Here, using a high-quality long-read assembly of T. cruzi, we performed comprehensive re-annotation and structural characterization of DGF-1. We identified seven tandemly repeated structural modules, here termed ribs, that form the extracellular region. This is followed by a distinct C-terminal membrane-associated region containing a conserved hat domain and multiple transmembrane helices. These domains are highly conserved across paralogous copies and the 7 ribs cluster according to positional identity rather than gene origin, indicating that the canonical seven-rib architecture predates the expansion of the family. DGF-1 proteins are present in the early-branching trypanosomatid species Paratrypanosoma confusum and several other trypanosomatids but absent in Leishmania and African trypanosomes, consistent with multiple independent secondary losses. Comparative analyses across Euglenozoa indicate that the DGF-1 architecture did not originate in free-living bodonids. Instead, Bodo saltans contains proteins with rib-like domains and others containing the membrane-associated region, whereas the first complete DGF-1 architecture appears in P. confusum. Phylogenetic and comparative genomic analyses indicate that the family subsequently underwent lineage-specific losses and independent expansions, the largest expansion occurring in T. cruzi. Together, these findings reconstruct the evolutionary emergence of the DGF-1 architecture from pre-existing structural modules and provide a framework for one of the largest and most enigmatic gene families in trypanosomatids.

Despite glutamates widespread role as the dominant excitatory transmitter in vertebrate brains, the early evolution of glutamate and its recruitment into neural signaling remain largely unknown. The major limitation is the lack of information on its distribution in early-branching basal metazoans, such as ctenophores (comb jellies). Here, using glutamate immunoreactivity (IR) in two ctenophore species with distinct ecologies (Pleurobrachia bachei and Beroe abyssicola), we show that glutamate IR is present in subpopulations of neurons within the subepithelial neural network and in small groups of mesogleal neuron-like cells, and that it differentially labels some muscle fibers. Remarkably, we also observed an enriched glutamate-ir signal within the nuclei of subepithelial neurons in Beroe. However, glutamate expression levels are species-specific, suggesting a tight coupling of glutamate recruitment for neural communication with energetic demands.

In the deep Pacific and Indian oceans, Alvinellid worms diversified about 100 million years ago to colonize a variety of hydrothermal vent environments. It has been suggested that the last common ancestor of this family was a thermophilic species. However, the evolutionary history of these worms is complex, with putative gene flow among ancestors. In this study, we investigated the early evolution of the family in relation to the diversification of thermal niches. We demonstrated that phylogenetic histories of alvinellid species greatly vary along chromosomes, possibly due to allele introgression between nascent ecotypes. We performed sequence reconstructions of ancestral cytosolic MDH and Cu/Zn SOD under the two most frequent phylogenies encountered along the genome. In silico simulations of folding stability for these two enzymes were highly correlated with their biophysical characterization (micro-calorimetry and differential scanning fluorimetry), and were used to predict the thermostability of thousands of alternatively reconstructed proteins. We further generalized the reconstruction at the proteome scale, taking the amino-acid usage bias as a proxy for folding stability in a new phylogenetic model accounting for amino-acid variations over time. Both approaches agree that the last common ancestors of Alvinellidae gained highly stable proteins, comparable to proteins of present-day thermotolerant species. Our computational protocol allowed us to validate this scenario under multiple phylogenetic hypotheses and different sequence reconstruction models. Considered together, Alvinellidae shed light on how metazoan species diversify in order to colonize different thermal niches, combining adaptive mutation and selection on genetic variants. SignificanceDeep-sea hydrothermal vents are among the most extreme environments on Earth. They, however, represent oases of life for a restricted number of highly specialized species. As such, Alvinellid worms constitute an exceptional family, including highly thermotolerant animals such as the Pompeii worm. Despite their vent endemicity, Alvinellidae thrive under contrasting ecological conditions, particularly regarding temperature. Understanding how this family diversified to colonize broad thermal regimes is of major importance for dissecting the mechanisms by which species adapt in highly unstable environments. Combining experimental protein resurrection, proteome-wide predictions, and new phylogenetic models, we assess the reliability of ancestral sequence reconstructions that represent molecular thermometers to evaluate past environmental conditions.

Understanding the underlying neuronal function in non-model organisms requires accurate resolution of gene structure and transcript diversity. Here, we present a comprehensive genome annotation tor the Jonah crab (Cancer borealis), a key experimental system in crustacean neurobiology, with a particular focus on transcriptome-supported neuronal gene architecture. By integrating long-read genome assembly with extensive transcriptomic evidence, we reconstructed gene models with high confidence, enabling detailed characterization of exon-intron organization, alternative splicing, and isotorm diversity across gene families. Functional classification revealed extensive representation of neural-associated gene classes, including ion channels and receptors, transporters, enzymes, zinc finger proteins, histones, structural proteins, and cell adhesion molecules, alongside a large set of previously uncharacterized genes. In this study we particularly focused on the neuronal and ion channel gene families known to underlie circuit-level neuronal function in C. borealis. We provide an in-depth analysis of 87 genes spanning 17 neural-related gene families and 41 neuropeptides, detailing chromosomal localization, gene length, exon-intron configuration, and transcript-supported isotorm structure. For many of these genes, transcriptomic data confirmed expression and refined coding boundaries. Comparisons with existing transcriptomic datasets demonstrate strong concordance in gene expression patterns while also revealing novel transcripts and expanded gene family members not previously annotated. Together, this genome and transcriptome-integrated annotation establishes a high-resolution framework tor studying neuronal gene organization in C. borealis. T his resource enables direct connections between gene architecture, transcript diversity, and neural function, supporting future investigations in crustacean neurogenomics, comparative genomics, and the evolution of nervous system complexity.

The ability to adapt shapes the future of any species or population. Adaptive potential is primarily determined by the spontaneous mutation rate {micro} and the mutation spectrum, which define the new genetic variation available to selection. Increasing evidence points to the plasticity of the mutation rate and spectrum, which vary according to environment. However, data is lacking for marine phytoplankton, an essential group for marine ecosystems as well as a major player in biogeochemical cycles. Here, we measure the plasticity of mutation rates and spectra in the chlorophyte Ostreococcus tauri by mutation accumulation experiments in 4 different conditions: low salinity, high salinity, low temperature, and high temperature. {micro}SNM is lowest under low salinity, doubles under high salinity and low temperature and quadruples under high temperature. SNM spectra become increasingly biased towards C:G[->]T:A transitions with increasing {micro}SNM. While {micro}ID does not change with the environment, the structural mutation rate {micro}SM is higher at low temperature, likely due to transposon activity. Given the rapid environmental changes associated with global climate change, mutation rates and spectra plasticity may critically influence the adaptive potential of marine primary producers.

Conserved synteny enables the reconstruction of ancestral chromosome organization across deeply diverged lineages, as demonstrated across metazoans. In Spiralia, comparative genomic analyses have suggested an ancestral karyotype comprising 20 chromosomes. We report high-quality genome assemblies for two Lima species (Lima caribaea and Lima lima) with 20 chromosomes and show that their chromosome organization matches this ancestral state, including four previously described fusion-with-mixing events. Interestingly, other Limidae genus did not retain the ancestral karyotype: they have undergone subsequent lineage-specific fusions. The conserved organization of Lima chromosomes provides a rare genomic anchor for reconstructing ancestral chromosome organization and lineage-specific chromosome evolution across Spiralia and, more broadly, Metazoa.

The twisted-wing parasites (order Strepsiptera) are well-known for their unusual life histories as obligate endoparasites of a wide variety of other insect orders. Here, we present the first high-quality genome assembly of a leafhopper-parasitizing strepsipteran species. We sequenced the genome of Elenchus koebelei using PacBio HiFi technology, yielding a primary assembly of 63.7Mb in 96 contigs, with an N50 of 1.60Mb and a BUSCO completeness score of 82.3%. In total, we recovered 8,167 protein-coding gene annotations, and 34.2% of the genome annotated as repeats. For the first time, we compare the genome content of three strepsipteran species and find that they are remarkably consistent in broad structure, with approximately 600 insect universal single-copy orthologs that appear to be completely missing from the order. These similarities imply a conserved genomic blueprint for strepsipteran parasitic strategy and/or differences that must be investigated at the post-translational level.

Amoebic gill disease (AGD), caused by the parasite Neoparamoeba perurans, is a major welfare and economic burden for the Atlantic salmon aquaculture industry. However, the spatial organisation of immune functions within the gill, the site of infection, remains poorly resolved. Here, we present the first spatial transcriptomic atlas of the Atlantic salmon gill affected by AGD, generated using 10x Genomics Visium on four AGD-positive fish, confirmed by gill scoring and quantitative PCR for parasite load. As no uninfected gills were included, this study maps the anatomical and immunological organisation of AGD-affected gills rather than defining AGD-specific pathology. Unsupervised clustering of spatial gene expression resolved distinct transcriptional domains that grouped into four principal anatomical compartments of the gill, including (i) epithelial and lamellar regions defined by keratin and osmoregulatory gene expression, (ii) gill arch regions enriched for connective tissue and smooth muscle markers, (iii) an erythroid-associated vascular cluster, and (iv) a skeletal muscle cluster with signatures of glycolysis and oxidative phosphorylation. Across these domains, we identified a spatially organised and diffused immune landscape, with pattern recognition receptors, antimicrobial effectors and chemokines concentrated at the filament surface, and immunoglobulin light chain transcripts consistent with B lymphocyte activity enriched toward the gill arch. Integration of the spatial data with a published single-nucleus reference atlas using reference-based deconvolution resolved the cellular identity underlying these domains and revealed frequent co-localisation of immune and structural cell types, suggesting regulated cell to cell communication within the gill-associated lymphoid tissue. Overall, our results show that the Atlantic salmon gill is not a transcriptionally uniform surface but a regionally specialised organ, with immune functions distributed across, and specialised within, distinct anatomical compartments

Sea urchins are pivotal models in cellular and developmental biology, but their biphasic life cycle with an extended larval life limits the ability to study post-metamorphosis and adult characters. Here, we introduce a genomically-enabled model system, the tuxedo urchin Mespilia globulus, which has rapid access to late life stages in sea urchins. We describe how we cultured M. globulus in a landlocked marine facility, raised larvae under artificial conditions and closed their life cycle. We established the experimental tractability of M. globulus: we labelled transcripts by hybridization chain reaction (HCR), and knocked out pigmentation genes to produce albino larvae using CRISPR/Cas9. We generated chromosome-scale genome assemblies for two individuals representing both sexes and two color morphs (red and blue), and compared the organisation of the 21 chromosomes of M. globulus with that of other camarodont echinoid models. We determined that M. globulus showed a conservative gene repertoire lacking the gene family expansions seen in other camarodont sea urchins. We annotated the complement of genes associated with pigmentation, immune and nervous systems and profiled their expressions in tissues and organs. Finally, we surveyed sex-related regions in genomes using genome assemblies and resequencing data, finding no evidence of heteromorphic sex chromosomes in M. globulus. Our findings highlight the accessibility of this new sea urchin model for studying the metamorphosis and adult biology of sea urchins.

The origin of multicellular animals from unicellular ancestors remains a major evolutionary question. To address this transition, a reconstruction of the nature of the last unicellular ancestor of animals (LUAA) is key, and requires broad sampling of extant unicellular relatives, which together with animals form the clade Holozoa. Environmental surveys suggest that unicellular Holozoa diversity extends beyond the five groups with characterized members (Choanoflagellatea, Ichthyosporea, Filasterea, Corallochytrea, and Atreyea). However, the extent of the environmental diversity within and outside these groups remains unclear. To fill this gap, we here systematically re-evaluated Holozoa diversity using environmental 18S rDNA sequences compiled from updated datasets, including long-read metabarcoding surveys. We report nine uncharacterized lineages inside Holozoa (seven newly identified here), provisionally placed outside the described groups. We also uncovered a previously unreported diverse lineage deeper inside Opisthokonta, separate from Holozoa and Fungi. In addition, we identified 21 uncharacterized lineages within Choanoflagellatea and Ichthyosporea (eleven newly reported here). We used short-read metabarcoding datasets to characterize the environmental and geographical distributions of these lineages. Many are globally distributed and some can reach relative abundances comparable to those of the described groups, while others are restricted to specific environments. Our findings reveal substantial uncharacterized diversity missing from the cultured taxa used in comparative studies of animal origins. By placing this diversity in a phylogenetic and environmental context, our study will guide the targeted isolation efforts needed to broaden the taxon sampling available to reconstruct LUAA and unravel the unicellular-to-multicellular transition that gave rise to animals.

Female choice is a central process of sexual selection that has shaped numerous phenotypes in animals. It may operate broadly across sexually reproducing organisms, including plants, not only because sex differences in sexual selection arise from anisogamy, wich refers to the unequal investment in gametes between sexes, but also because it can emerge from simple variation in female reproductive morphology. Here, we provide the first empirical test of cryptic female choice in an angiosperm by examining whether pollen and pistil traits jointly influence paternal fertilization success. Using experimental pollen competition, paternity analyses, and trait measurements, we show that pistil traits can bias paternity toward pollen donors with specific pollen traits. These results demonstrate that cryptic female choice operates in plants, paralleling mechanisms described in animals.

RNA interference (RNAi) offers a sequence-specific approach to pest control. In insects, Dicer-2 processes double-stranded RNA (dsRNA) into small interfering RNA (siRNA) duplexes, from which the RNA-induced silencing complex (RISC) retains a guide strand. Only antisense-loaded RISC can mediate cleavage of the target transcript. However, how sequence features shape the RISC-bound siRNA pool in pests remains poorly understood, limiting opportunities for sequence optimization. Here, we profiled RISC-bound siRNAs following injection of 34 insecticidal dsRNAs targeting 11 essential genes in Tribolium castaneum larvae. We computationally reconstructed 7,879 siRNA pairs and examined associations between sequence features and strand bias. Differences in GC identity at terminal paired positions 1 to 5, used as a proxy for local thermodynamic asymmetry, correlated with strand bias, with the strongest correlations at the first two paired positions. ORF targeting and reduced predicted antisense self-folding were also associated with higher antisense fractions. Analysis of non-templated terminal additions revealed predominantly 3-prime uridylation, a known signature of small RNA turnover, along with putative 3-prime trimming. Among ORF-associated siRNA pairs, sense strands showed higher relative U-tailing abundance, based on 3-prime uridylated and putatively trimmed-and-3-prime-uridylated reads relative to perfect 21-nt reads, than antisense strands. Antisense strands with the least predicted self-folding also showed low relative U-tailing abundance. These observations are consistent with sequence-dependent contributions from both guide-strand selection and differential post-RISC-loading siRNA retention, although a causal link remains to be established. The identified associations provide a basis for testing whether dsRNA sequence optimization can improve pest control efficacy and reduce off-target activity.

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.

Background: The insect cuticle is a chitin-containing extracellular matrix whose formation requires precise coordination of chitin biosynthesis, fibril organization, and post-synthetic maturation. Although lipids are essential components of the cuticle, the molecular mechanisms linking lipid transport to chitin deposition remain poorly understood. Here we identify TcFabp8, a fatty acid-binding protein in Tribolium castaneum, as a functionally important regulator of chitin synthase 1 (Chs1)-dependent cuticle formation. Results: RNAi-mediated depletion of TcFABP8 caused molting defects, reduced cuticle thickness, loss of apical TcChs1 immunoreactivity, and reduced chitin deposition and organization. The finding that TcCHS1 transcript levels were unaffected by RNAi targeting TcFABP8 suggests that a post-transcriptional step, potentially involving vesicular TcChs1 transport, is affected, thereby impairing chitin synthesis and deposition. Synchrotron X-ray diffraction analysis of adult elytra revealed that TcFabp8 depletion preserved characteristic -chitin-associated scattering features, but altered chitin fibril accumulation, crystalline coherence, and the spatially ordered alignment of fibrils within the cuticular matrix. The TcFabp8 gene locus encodes splice variants yielding different N-terminal architectures: TcFabp8X1 carries a Sec/SPI signal peptide which is absent in TcFabp8X2 and TcFabp8X3. Immunocytochemistry using TcFabp8-specific antibodies detected intracellular signals in subapical cytoplasmic puncta and extracellular signals within the cuticle layers, which partially colocalized with Nile red fluorescence indicating lipid-rich environments. Conclusions: Together, our data demonstrate that TcFabp8 is a critical facilitator of TcChs1-dependent chitin deposition and extends functional connection between lipid transport and chitin biosynthesis in insects.