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.

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

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.

Holocentric chromosomes allow rapid genome changes through chromosomal rearrangements such as fissions, fusions, inversions or translocations. The plant genus Carex shows one of the highest rates of karyotypic evolution among holocentric organisms. We studied the genomic patterns underlying chromosomal rearrangements in the karyotypic radiation of the narrow endemic species Carex helodes (2n = 68-75). Comparing genome assemblies of C. helodes from the two karyologically distinct extremes of its European distribution, revealed a striking number of eight chromosomal rearrangements including fusions, translocations and inversions. Genomic breakpoints are gene-poor and TE-rich, corroborating findings in other species and suggesting common genomic characteristics that facilitate the evolution and establishment of chromosomal rearrangements. We identified a chromosomal inversion exhibiting patterns of purifying selection and enrichment in functional genes that potentially mediate rearrangement tolerance. Conversely, another inversion displayed elevated sequence divergence and enrichment in response to temperature stress and phosphate limitation, matching key environmental variables that differ between the study localities. The establishment of chromosomal rearrangements along Carex helodes European populations was likely driven by demographic bottlenecks and distinct genomic features at breakpoints. Our findings provide preliminary evidence on the rearrangement role in population differentiation either as reproductive barriers or as genomic islands of differentiation.

Marine photosynthetic microbes contribute substantially to global primary production, yet many algal lineages still lack reference genomes with the continuity and annotation quality required for fine-scale structural, regulatory and comparative analyses. Ostreococcus tauri, one of the smallest known free-living photosynthetic eukaryotes, has been a model marine picoeukaryote for over two decades. Despite successive improvements to its historical reference genome, previous assemblies retained hundreds of gaps and incomplete genes, hampering high-resolution genomic analyses. Here, we present O. tauri RCC4221 genome version 2026, a telomere-to-telomere assembly of all 20 chromosomes spanning 13.34 Mb with no gaps. This assembly combines PacBio long-read sequencing, Illumina short-read polishing, correction of unresolved regions guided by independent Nanopore-based assemblies. The updated reference supports a curated annotation comprising 7,683 protein-coding genes, 48 tRNA genes, 3 rRNA operons, 116 medium-sized noncoding RNAs, one signal recognition particle RNA and 138 small nucleolar RNAs. It also improves gene-model integrity and recovers candidate coding loci absent from the 2014 reference. Structural analyses resolved the organization of the two atypical low-GC chromosome 2 and 19 that contain duplicated regions that were collapsed or misrepresented in previous assemblies. Finally, bisulfite sequencing and PacBio SMRT sequencing revealed a dual DNA methylation landscape, with CG-context cytosine methylation concentrated in gene bodies and N6-methyladenosine (m6A) enriched at the start codon. The updated O. tauri 2026 assembly provides a complete and curated reference resource for chromosome biology, comparative genomics, epigenomics and RNA biology in a model marine picoeukaryote.

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

The NAD+-dependent histone deacetylase Sirt6 regulates transcription of multiple classes of genes, including those involved in metabolism, immune response, oxidative stress response, and development. Defining the Sirt6-regulated transcriptome is relevant to understanding the various important physiological roles of Sirt6, such as extending lifespan, maintaining metabolic health, and tumor suppression. Numerous studies have identified Sirt6 target genes, using both targeted and genome-wide approaches; however, consensus is limited and there has yet to be a systematic analysis of gene expression changes induced by altering Sirt6 levels. In the present study, we conducted a meta-analysis of 19 mammalian RNA-seq datasets in which Sirt6 levels were perturbed (knockout, knockdown, or overexpression). Our analyses included Gene Set Enrichment Analysis, pathway analysis of differentially expressed genes, and identification of individual differentially expressed genes. Our analysis identified consistent gene expression changes associated with lowering Sirt6 levels, including increased expression of immune response and ribosomal protein genes and reduced expression of lipid oxidation and oxidative phosphorylation genes. Extracellular Matrix and E2F target genes also had consistently increased expression upon Sirt6 reduction, highlighting novel regulation by Sirt6. To determine the conservation of gene regulation by Sirt6, we performed additional RNA-Seq meta-analysis on tissues from Drosophila melanogaster with Sirt6 deletion and overexpression. The fly datasets produced similar results to the mammal results, except for lipid oxidation genes, which were found to increase in Sirt6-low conditions. These results provide consensus about conserved and novel pathways transcriptionally regulated by Sirt6.

Although it is well established that certain stages of development are molecularly more conserved than others, the reasons for this phenomenon remain largely unknown. We study molecular conservation in the development of an organ, the molar, by comparing the temporal profiles of expression in mice and hamsters. We find that the cause of conservation of expression and of coding sequences changes over molar development. Gene expression levels display a classical increase of divergence as development progresses. In terms of genes expressed, the composition of early and late stages is better conserved and enriched in pleiotropic genes, yet each stage mobilizes different sets of pleiotropic genes, cell division for bud growth and secretion for tooth mineralization. Moreover similar patterns of higher divergence of gene sets and of coding sequences at mid development, are caused by different biological phenomena, in that case heterochronies and blood colonisation respectively. In conclusion, the patterns of molecular conservation in developing molars are shaped by a combination of processes intrinsic to the teeth, and by negative and positive selection on functions which are mostly extrinsic to the teeth. This is likely translatable to explain molecular conservation patterns in many other biological systems. AUTHOR SUMMARYFor species to evolve different adaptations to different life styles, their anatomy has to evolve correspondingly. This in turn implies evolution of the embryonic development of anatomical structures. Notably, tooth shape can evolve rapidly as an adaptation to different diets. Mice and hamsters are closely related rodents who yet differ in the shape of their molars, and thus in their development. In this study, we investigated why the genes active in molar development are more or less similar between the two species from early tooth bud to fully formed embryo molar. We found that early and late molar development were slow evolving, while mid-development was evolving faster. But surprisingly, this was in part due not to tooth evolution, but to the involvement of genes which are active in other processes in the body. For example an influx of immune cells also brings fast evolving immune genes. This helps us understand better the complexity of causes of apparently simple evolutionary patterns.

Drosophila RNA viruses often persist in wild and lab populations, yet their tissue and cellular tropism is poorly understood. In the Fly Cell Atlas (a comprehensive Drosophila single-nucleus transcriptome) data, we detected four RNA virus infections: Nora virus, Drosophila A virus, Drosophila C virus, and Newfield virus. Nora and Drosophila A virus were the most abundant and widespread across tissues and cell types, while Drosophila C virus and Newfield virus RNA transcript were only found in oenocyte and fat body tissues. We found transcriptional changes associated with viral infection in canonical viral immunity genes (e.g. Vago, vir-1). Additionally, we observed that during persistent viral infections, transposable element (TE) transcripts were upregulated in somatic cells. TEs are traditionally associated with the germline, but recent studies and our data suggest they are also expressed in somatic cells. Using the Fly Cell Atlas data, we found that distinct somatic cell types express specific TE subtypes, indicating regulated and cell-type specific TE activity often overlooked in transcriptomic studies. We present Fly Viral Atlas (https://flyviralatlas.shinyapps.io/home/), a single-nucleus level atlas of RNA viruses and TE expressions in Drosophila, providing new insights into viral tropism and TE dynamics across cell types and tissues.

Parental care is widespread across the animal kingdom and plays a critical role in offspring development. Yet, its broader genetic and evolutionary impacts remain underexplored. Here, using the biparental burying beetle Nicrophorus vespilloides as a model, we show that parental care acts as a genetic capacitor: it allows genetic variation to accumulate while care is present and releases it when care is disrupted. By experimentally manipulating care, we demonstrate that parental care suppresses genetic variation associated with offspring body size, which is released when care is lost. To investigate the underlying molecular mechanisms, we generate a chromosome-scale genome assembly for N. vespilloides, alongside a single-nucleus gene expression atlas and epigenomic datasets from larvae reared with and without parental care. We find that the loss of parental care induces molecular stress, disrupting the expression of the protein chaperone Hsp83, which is a well-known molecular capacitor, alongside other putative mRNA chaperones. Moreover, our results suggest that parental care buffers development by maintaining an open, responsive chromatin landscape and redundant gene regulatory interactions. Overall, our work reveals that parental care shapes the storage, expression and release of genetic variation with broad implications for adaptation and evolution.

Sensory systems, built around specialized cell types, are central to how animals perceive and respond to their environments. Yet many of the molecular components defining these systems predate the origin of animal multicellularity. Among these, transient receptor potential (TRP) channels form a polymodal and evolutionarily ancient superfamily of ion channels involved in diverse sensory processes. To better understand how sensory complexity emerged in animals, we investigated the diversity of TRP channels in choanoflagellates, the closest living relatives of animals. Using a combination of homology-based searches, phylogenetics, and structural predictions, we find extensive TRP channel repertoires across choanoflagellates, including representatives of most major animal TRP channel families. Comparative analyses across species revealed two distinct evolutionary patterns for TRP channel families: conserved, low-copy families with stable domain architectures, and lineage-specific expansions within the families TRPM and TRPW, indicative of functional diversification. Functional insights from fluorescent localization studies in the choanoflagellate Salpingoeca rosetta demonstrated that TRPA, TRPC, and TRPV channels are spatially segregated within the collar complex, a key interface for environmental sensing and feeding. Distinct localization domains, along with evidence for heteromeric interactions between TRPA paralogs, suggest that subcellular organization likely contributes to sensory specialization in these single cells. Together, our findings indicate that a diverse and functionally versatile TRP channel toolkit was already present in the last common ancestor of choanoflagellates and animals. We propose that the evolution of animal sensory systems involved both expansion and reorganization of this ancestral repertoire, with subcellular patterning in unicellular organisms representing a precursor to cell-type specialization in multicellular animals.

BackgroundThe black soldier fly (Hermetia illucens) is an emerging model for bioconversion and industrial rearing. Its genome is highly repetitive, yet the contribution of transposable elements (TEs) to population divergence and demographic processes. The sampled populations represent a gradient of demographic histories, including wild and near-wild North American populations, and domesticated European strains with shared industrial origins. Difference in TE composition may influence genome structure, regulatory variation, and evolutionary responses to captive environments. ResultsA comparative analysis of the repetitive landscape was done for four H. illucens genomes, one of which is a wild-caught specimen. Total repeat content was high across all assemblies (67.6% to 70.8%) and dominated by LINE elements. Class-level TE diversity was nearly identical among genomes, but multiple DNA transposon families showed distinct lineage-specific differences. Large families including Maverick and Academ were generally depleted relative to the wild sample. Divergence profiles revealed patterns consistent with recent turnover in several families. Family level turnover, rather than class level change, accounted for the most difference among the genomes. TE-associated structural variants (TESVs) were also not uniformly distributed. Most chromosomes showed mid-chromosome enrichment, and a pronounced TESV peak on chromosome 5 overlapped a histone rich region containing many unclassified repeats. Use of a repeat library derived from multiple genomes increased the number of detected TESVs and improved classification within complex regions, demonstrating that multi-genome libraries enhance annotation accuracy compared to single reference-based models. ConclusionsMultiple DNA transposon families show evidence of recent or lineage-specific amplification in H. illucens, suggesting that TE amplification contributes to genome variation during demography-associated TE turnover. The multi-genome-based library improved TE detection and classification, providing a proof of concept that even a small lineage-inclusive repeat library enhances annotation accuracy and capture TE diversity missed by single-reference approaches. Together, these findings demonstrate that TE family turnover plays a significant role in shaping genome architecture and adaptation in this species.

Epithelial cells in Hydra perform an unusual combination of functions: they divide continuously like adult stem cells while simultaneously executing the complex physiological tasks of differentiated epithelia. This challenges the traditional distinction between proliferative stem cells and terminally differentiated tissue, raising the question of how a single cell type integrates these opposing roles. Using electron microscopy, we examined morphological characteristics that define the stem-like and differentiated states of Hydras ectodermal and endodermal epithelial cells. Stemness is reflected by nuclear characteristics of active proliferation, including extensive euchromatin, large nucleoli, and the presence of nuage. However, differentiated epithelial cells exhibit strong apical-basal polarity, various endomembrane compartments for endocytosis and transport, specialized secretion mechanisms, and basal muscle processes with dense-core vesicles implicated in hormonal communication. Cryofixation improved ultrastructure preservation, elucidating the pleiomorphic configurations of complex intracellular channel systems traditionally presenting as singular vacuoles. This may shed new light on possible functions of this compartment. Taken together, Hydra epithelial cells combine ancient stem cell traits with highly specialized differentiated functions. This multifunctionality provides insight into the cellular organization of early-branching animals and suggests that multifunctional epithelia may represent an ancestral condition preceding the strict segregation of stem and differentiated cell lineages in bilaterians.

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.

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.

Rafflesiaceae, known for producing the largest flowers in the world, are obligate parasites that exclusively infect Tetrastigma sp. (Vitaceae). Despite their unique biology, the interactions between parasitic tissues and host roots remain poorly understood, particularly during the flower morphogenesis phase, where parasitic tissue erupts through the host root. Here, we performed dual transcriptome analyses of two Rafflesiaceae species and their respective Tetrastigma hosts: Sapria himalayana with T. cauliflorum and Rafflesia speciosa with T. magnum. Our findings reveal species-specific transcriptional responses in Tetrastigma, suggesting divergent parasitism strategies between Rafflesia and Sapria. Moreover, we identify molecular signatures of parasitism that parallel plant gall formation, particularly in genes governing cell wall modification and host tissue reorganization. Unlike bacterial or insect-induced galls, these mechanisms may involve fungal symbionts, highlighting the unique nature of these interactions. Together, our results demonstrate that Rafflesiaceae parasitism represents a complex tripartite relationship among host, holoparasite, and associated microbes, offering new insights into the hidden biology of these remarkable parasitic plants.

Single-cell RNA sequencing technology dramatically changed the way we investigate transcriptomes. However, the amount and complexity of data generated by such methods poses new challenges for biologists who are trying to extract detailed insights into the genetic programs that drive cellular functions and differentiation. To provide a more intuitive understanding of cell specific gene expression programs, we developed a novel approach for exploiting scRNA-seq data that detects individual gene expression levels in each cell, by avoiding dimensional reduction methods. This was achieved by focusing our analysis on individual cells with a high sequencing coverage (above 15000 Unique Molecular Identifiers (UMIs)). Such High Coverage Cells (HCC), were found in all five C. elegans scRNA-seq datasets we investigated and constitute direct quantitative experimental observations of the mRNA content of individual cells. Clustering the complete gene expression matrix for these cells, we identified gene sets specific for most C. elegans tissues. Among each set we found genes that are dominating cell specific transcriptomes as well as genes that are restricted to particular cell types but are a thousand fold less expressed. For each cell type or subtype we characterized, we identified a set of genes with expression restricted to those cells that were not previously associated with the corresponding tissue. Our results demonstrate that by focusing on HCCs, we can provide high-resolution quantitative descriptions of cellular expression landscapes that are immediately exploitable for researchers to generate new biological hypotheses. Overall, we demonstrate that HCCs represent a powerful and largely unexplored source of biological insights and suggest that future scRNA-seq experiments could benefit from focusing on HCC enrichment to capture and exploit the full complexity of cellular transcriptomes.

Hearing in Lepidoptera mediates a range of ecologically important behaviours, including mate communication, predator avoidance, and acoustic signalling. In moths, the evolution of predator-prey interactions with bats has further shaped hearing through a sensory arms race, with repeated co-option of auditory organs to detect and evade echolocating predators. Despite significant prior characterization of the neurophysiology and behaviour of hearing in moths, the genetic basis of hearing is poorly understood in most insects. In this study, we identify a core set of putative auditory genes in Lepidoptera using a combination of homology-based searches from Drosophila and evolutionary rate analyses. We find 56 genes present across all species and investigate whether gene copy number varies among non-hearing and hearing lineages and among 3 different ear types. We discovered seven genes associated with ear type and one with ear presence, but did not find significant losses in gene copy number in non-hearing species. We identified three genes (btv, Dnai2, and nompB) with strong evidence of selection in hearing clades and five genes with weaker evidence of selection. We discuss the potential roles of btv, nompB, and Dnai2 in ciliary transport and the aging of hair cells, as well as the possibility of actively amplified hearing. Our study serves as a primer and resource for further gene mining and functional testing of auditory genes in moths and other insects.

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.

Trehalose is a primary circulating sugar in insects and essential for energy homeostasis, yet its non-coding RNA-based regulatory circuitry remains enigmatic. Here, we characterize a conserved long non-coding RNA, lncRNA1, as a post-transcriptional regulator of trehalose-glucose homeostasis in Lepidoptera. lncRNA1 encodes a structurally stable, pseudoknot-containing transcript that is strongly induced upon trehalose pathway perturbation and exhibits a reciprocal developmental expression pattern relative to the trehalose metabolism enzymes. RNAi-mediated silencing of lncRNA1 in Helicoverpa armigera elevates TPS/TPP and Treh transcript abundance, increases enzyme activities, reduces haemolymph trehalose, raises glucose. This drives broad transcriptomic and metabolomic reprogramming of carbohydrate, lipid, and growth-signalling pathways, resulting in accelerated larval growth. Overexpression of lncRNA1 reverses these phenotypes. Mechanistically, lncRNA1 physically associates with TPS/TPP and Treh mRNAs through evolutionarily conserved sequence motifs, modulating their post-transcriptional dynamics. Targeted deletion of these motifs abolishes regulatory activity and disrupts metabolic homeostasis. This regulatory axis is functionally conserved in Spodoptera frugiperda, validated across loss-of-function, gain-of-function, and cell-based systems. Our findings reveal a conserved lncRNA-based layer of post-transcriptional control over insect energy metabolism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/738560v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@f1c170org.highwire.dtl.DTLVardef@1d2a9c7org.highwire.dtl.DTLVardef@1886786org.highwire.dtl.DTLVardef@44b83b_HPS_FORMAT_FIGEXP M_FIG C_FIG