Museum specimens are widely used for PCR-based pathogen detection, yet their potential for metagenomic discovery of beneficial microbes remains underexplored, largely due to difficulties in distinguishing true symbionts from contaminants. Here, we use metagenomics of museum specimens to uncover symbioses in endangered or difficult-to-collect animals, such as nudibranchs. To date, Doriopsilla is the only nudibranch demonstrated to harbor an uncultured symbiont involved in chemical defense, leaving it unclear whether comparable associations occur in other nudibranchs. We hypothesized that bona fide symbionts should belong to abundant, uncultured lineages consistently present across individuals of the same host taxon collected across space and time. Using ethanol-preserved specimens archived for up to 30years, we doubled the number of available nudibranch microbiome datasets and found that dominant uncultured symbionts are rare, with most nudibranchs likely relying on alternative chemical defense mechanisms. An exception were Polycera and Felimare that contained two previously unknown symbionts, Candidatus Polyceribacter and Candidatus Felimaribacter, from distinct uncultured orders that are globally rare in marine metagenomes. These symbionts encode diverse biosynthetic gene clusters exhibiting strain- and species-level microdiversity consistent with metabolites previously reported from their hosts. Their restricted host distribution, phylogenetic distinctiveness, and phylogenetic similarity to symbionts of sponges or corals that are not nudibranch prey, support long-term evolutionary specialization and functional convergence. Fine-scale diversification further suggests host-driven microbial adaptation following symbiosis establishment. Overall, this study establishes museomics as a robust framework for symbiosis research and advances understanding of the evolutionary and chemical ecology of host-microbe interactions in rare marine invertebrates. Video Abstract.

Elcysma westwoodi is a major pest of cherry trees (Rosaceae), which contain high levels of cyanogenic glucosides that release toxic hydrogen cyanide upon tissue damage. To investigate the genomic basis of host-plant cyanogenic glucoside detoxification and the evolution of chemical defence genes, we generated a PacBio HiFi-based genome assembly of E. westwoodi. The final assembly spans 460.62 Mb with high contiguity (contig N50 > 15 Mb) and near-complete recovery of conserved genes (99.6% BUSCO completeness). We predicted 19,502 protein coding genes, with annotation completeness reaching 95.2% BUSCO. Comparative analyses of three Zygaenidae genomes revealed strong conservation of chromosomal synteny and showed that E. westwoodi shares a more similar detoxification gene repertoire with Achelura yunnanensis, another Rosaceae feeder, than with the more divergent, non-Rosaceae feeding Zygaena filipendulae. Genome-wide analysis identified 272 detoxification-related genes, indicating substantial capacity for xenobiotic metabolism. Phylogenetic analysis supported long-term conservation of a vertically inherited, single-copy β cyanoalanine synthase (bCAS) gene involved in cyanide detoxification. In addition, structural and phylogenetic analyses revealed parallel retention of CYP405A and CYP332A in the two Rosaceae feeding species, a pattern most parsimoniously explained by the inheritance of an ancestral cyanogenic toolkit rather than by independent convergent recruitment, whereas UGT33A retained the canonical GT-B fold characteristic of UDP glycosyltransferases, indicating the evolutionary stability of phase II conjugation enzymes. Together, these findings indicate that adaptation in E. westwoodi proceeds through targeted gene-level evolution within an otherwise conserved genomic framework.

Rapid ecological speciation in gall inducers.

Seasonal rainfall shapes biological responses in tropical ecosystems, yet how tropical organisms integrate behavioral and physiological responses to cope with seasonality remains poorly understood. We assessed how four poison frog species with contrasting reproductive strategies respond to dry and wet season environmental conditions. We quantified spatial behavior, microhabitat use, hormone release rates, and chemical defenses in two seasonal breeders (Allobates femoralis and Ameerega trivittata) and two year-round breeders (Ameerega macero and Ameerega shihuemoy). Seasonal breeders exhibited pronounced sex-specific shifts in space use, where males expanded their space use during the wet season, likely to track reproductive opportunities, while A. femoralis females increased their spatial use during the dry season, likely responding to foraging demands when prey resources are sparse. Year-round breeders maintained similar space use across seasons, likely reflecting their ability to access key resources within the same space to reproduce year-round. Microhabitat use was flexible, as seasonal breeders shifted toward humid refugia during the dry season and reproduction-associated microhabitats during the wet season, whereas year-round breeders selected microhabitats that facilitate continuous reproduction across seasons. Despite these behavioral responses, water-borne corticosterone and testosterone, as well as chemical defenses, showed no consistent seasonal variation, suggesting that behavioral responses may be partially decoupled from shifts in endocrine and chemical defenses. These results support a role for behavioral buffering in mediating responses to seasonal environmental variation. However, given the increasing unpredictability in rainfall timing and intensity as a result of climate change, how these coping strategies will function in the long term is uncertain.

Terpenes, abundant in pine trees, function as chemical defenses against pathogens such as the pinewood nematode, the agent of destructive pine wilt disease (PWD). Paradoxically, certain terpenes like α-pinene and β-pinene are known to enhance pinewood nematode population growth; however, the mechanisms behind this adaptation remain unclear. This study aims to investigate how terpenes influence nematode physiology, resource allocation, and pathogenicity. High terpene concentrations (137.6 mg/mL), particularly the 10:8 α-pinene/β-pinene ratio prevalent in pinewood nematode-infected pines, enhance nematode population growth and thus pathogenicity by redirecting lipid reserves toward reproduction, while depleting resources for body size and shortening lifespan. Transcriptomic analyses reveal that terpenes up-regulate genes in lysosomal pathways. Pharmacological inhibition of lysosomes and RNA interference knockdown of the lysosomal regulator HLH-30/TFEB suppress reproduction and extend lifespan, suggesting key roles of lysosomal signaling in the trade-off between reproduction and lifespan. Pinewood nematode co-opts host defensive compounds as signals, triggering a lysosome-mediated shift in resource allocation that favors rapid population expansion and enhanced pathogenicity at the expense of individual longevity. Targeting lysosomal signaling or terpene biosynthesis could disrupt nematode adaptation, offering innovative strategies for sustainable PWD management. © 2026 Society of Chemical Industry.

Plant hybridization often alters herbivore-relevant traits, yet the effects of hybrid feeding on specialist herbivores, especially those that co-opt host-plant defenses, are largely unknown. We examined how naturally derived hybrids between Asclepias syriaca and A. speciosa ( A. syriaca x speciosa ) affect monarch butterfly ( Danaus plexippus ) performance and cardenolide sequestration. Across three independent greenhouse experiments, monarch caterpillars reared on genetically heterogeneous A. syriaca x speciosa hybrid milkweeds grew more slowly than caterpillars reared on either parental species. Monarchs reared on hybrids also, on average, sequestered lower total cardenolide concentrations into adult tissues. However, plant measures of cardenolide chemistry did not differ significantly among parental and hybrid host groups, and measured physical defenses (trichome and latex) showed only partial differences among groups, and none in the expected direction. Element analyses of plant leaf tissue, although preliminary, revealed a marginally significant negative relationship between caterpillar growth and manganese level, with hybrids containing between two- to three-fold higher levels of manganese as either parental species. These results indicate that host-plant hybridization can alter both specialist herbivore performance and acquired chemical defense, even when broad measures of host defenses do not explain those effects. Hybridization may therefore influence plant-herbivore interactions not only by changing host quality, but also by altering how specialist herbivores acquire plant-derived defenses.

Chemical defense: Plants play CD and pathogens face the music.

The reciprocal evolutionary processes among phytophagous insects and host plants involve host shift in the herbivorous insects in response to defense chemicals, such as plant-derived terpenoid compounds that comprise a key element for adaptive radiation, leading to chemosensory diversification. To reveal the multimodal chemosensory targets mediating avoidance behavior of insects in response to irritant stimuli, in this study, we explored multimodal target molecules toward (-)-α-thujone, a bicyclic monoterpenoid compound, focusing on the red flour beetle, Tribolium castaneum. The area-preference test revealed significant avoidance behavior in adult T. castaneum toward (-)-α-thujone. We applied systemic RNAi methodology to knock down hypothesized targets; knocking down the resistance to a dieldrin (Rdl) subunit comprising a γ-aminobutyric acid (GABA)-activated receptor significantly suppressed the avoidance behavior toward (-)-α-thujone, rather than l-menthol. Avoidance behaviors were significantly suppressed after knocking down thermo (TcTRPM) and non-thermo (TcTRPL) TRP channels. Subsequently, quantitative reverse transcription (qRT)-PCR-based spatial expression analysis of TcRdl and TcTRPL revealed high TcRdl transcript expression in the head, followed by the antenna, and prominent TcTRPL transcript expression in the head. Furthermore, the reduced avoidance behavior in antenna-dissected and knockdown of the olfactory receptor coreceptor (TcOrco) lines confirmed the involvement of antenna in the underlying mechanism. The results reflected potential molecular targets including non-thermo TRP channel involved in (-)-α-thujone-mediated avoidance behavior in T. castaneum and indicated multimodality, as exhibited by N,N-diethyl-3-methylbenzamide (DEET), the gold standard of repellent, providing useful insight for further research on the development of new repellents and excito-repellents. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that serve as defense compounds in Arabidopsis and other members of the Brassicales. Although the enzymatic pathway that produces GSLs is well-studied, the upstream mechanisms that control their tissue-specific synthesis are poorly understood. We identified a novel repression module that transcriptionally regulates GSL levels in sepals, the modified leaves that protect reproductive tissues within the floral bud. GLABRA2 (GL2) INTERACTING REPRESSOR1 (GIR1) interacts directly withArabidopsis thalianaMERISTEM LAYER1 (ATML1), an HD-Zip IV transcription factor known to be required for giant cell formation in the sepal epidermis. This interaction requires a predicted Zn finger of GIR1 and the C-terminal START adjacent domain (STAD) of ATML1. Thegir1loss-of-function mutants exhibit excess giant cells, in contrast toatml1mutants which display fewer giant cells, supporting the role of GIR1 as a negative regulator of ATML1. We confirmed that GIR1 interacts with TOPLESS (TPL) and TOPLESS-RELATED (TPR) corepressors, and coimmunoprecipitation demonstrated that GIR1 acts as an adaptor protein connecting ATML1 and TPL/TPR. RNA sequencing revealed that numerous genes involved in GSL biosynthesis, including the key transcriptional regulator MYB29, are upregulated ingir1mutants. Consistent with the transcriptomic data, chemical analysis revealed thatgir1mutants display elevated GSL levels in sepals. Mass spectrometry imaging confirmed high GSL accumulation ingir1sepals compared to wild type andatml1. Overall, our findings uncover a previously unrecognized link between cell expansion and GSL metabolism, suggesting strategies for engineering plants with cell-type specific GSL profiles.Significance StatementPlants belonging to the order Brassicales produce sulfur-containing glucosinolate (GSL) metabolites that serve in defense against herbivory. In cruciferous vegetables such as broccoli and kale, these compounds contribute to their unique flavors and health-promoting attributes. In agriculturally important oilseed crops, they affect the palatability of animal feeds. Here, we identified a novel transcriptional regulatory module that controls GSL biosynthesis in the epidermis of the sepal, the floral organ that protects the reproductive tissues. This regulatory module also controls cell expansion of specific cell types in the sepal, demonstrating a surprising connection between cell growth and a chemical defense pathway in plants. Our results suggest strategies for engineering crops with tissue-specific GSL profiles to fit agronomic needs.

Bacteria deploy diverse antiphage defense systems, including small bioactive molecules providing protection at the multicellular level. DNA-intercalating anthracyclines, such as daunorubicin, exhibit broad antiphage activity, but the underlying mechanism has remained elusive. Here, we systematically screened the Escherichia coli BASEL phage collection to elucidate the mode of action of DNA-intercalating antiphage molecules. We identified taxonomically distinct clusters of susceptible viral groups and show that for the Tequintavirus Bas33 (Markadamsvirinae), daunorubicin blocks infection after first-step transfer. In the presence of daunorubicin, continued expression of pre-early genes leads to abortive infection via "mutual destruction," where both phage and host succumb. Analogous abortive-infection phenotypes occur across taxonomically diverse phages exposed to chemically distinct DNA-intercalating molecules. Notably, we show that daunorubicin synergizes with downstream nucleic acid-targeting defenses underscoring context-dependent outcomes. Together, these findings reveal how chemical defense contributes to the multilayered antiviral immunity and highlight the intricate interplay between mechanistic inhibition and infection outcome.

Black rot, caused by Phytophthora parasitica, poses a significant threat to the commercial cultivation of Dendrobium spp., particularly the cultivar 'Earsakul'. However, the black rot resistance mechanisms in Dendrobium Sonia 'Earsakul' remain unexplored. This study elucidates the molecular mechanisms underlying black rot resistance by comparing a resistant ethyl methanesulfonate (EMS)-mutagenized line, SUT13E18301 with a susceptible non-mutagenized control, SUT16C014 in control (without pathogen inoculation) and inoculated conditions. Resistance phenotyping using detached leaf assay revealed stark contrasts in disease severity, prompting comprehensive transcriptomic analyses via RNA sequencing at 12- and 24-hours post-inoculation (hpi), compared with control condition. Bioinformatic analysis identified 4,190 significantly differentially expressed genes (DEGs), most of which were associated with defense responses. At the early stage (12 hpi), the genes related to signaling pathway, transcriptional reprogramming, and cell wall modification were differentially expressed. At the later infection stage (24 hpi), results highlighted genes with important roles in pathogen recognition, hormone signaling, cell wall modification, antimicrobial compounds/defense proteins production, hypersensitive response, and detoxification, all related to disease resistance mechanisms. Among these, nearly all of twenty-three candidate genes, including peroxidase (POD) 51-like, beta-glucosidase 11-like, pectinesterase, chitinase 2-like, transcription factor MYB6, and fasciclin-like arabinogalactan protein 11 (FLA11) showed up-regulation in the resistant line, whereas reduced responses in the susceptible line. Quantitative real-time PCR (qPCR) validation confirmed these expression patterns, showing strong positive correlation (R = 0.94). Furthermore, a highly significant increase in POD activity was observed at 24 hpi compared to 0 and 12 hpi in the resistant line. Conversely, the susceptible line showed no significant differences across the time points. Additionally, the weighted gene co-expression network analysis (WGCNA) and the regulatory network of candidate genes indicated that resistance is mediated by complex interactions among multiple regulatory components, rather than a single predominant pathway, and integration of structural and chemical defenses. These findings provide crucial insights into the genetic basis of black rot resistance and identify valuable molecular targets for breeding black rot resistance inDendrobium.

In coastal ecosystems, chemically rich species like gorgonians rely on specialized metabolites and symbiotic microbes for health and defense. In the Southwestern Atlantic, the elephant ear coral Phyllogorgia dilatata builds structurally complex forests and provides habitat for several species. Recent declines in cover have been linked to widespread disease, fouling, and necrosis. The loss of chemical defense due to anthropic perturbation has never been reported in the marine environment. We investigated whether pollution-driven stress could lead to a dysfunctional holobiont and impairment of its chemical defense. Using chromatography coupled to high-resolution mass spectrometry and molecular networking, we profiled secondary metabolites and used 16S rRNA gene amplicon sequencing to characterize microbial communities, relating these data to visual surveys of P. dilatata gorgonian forests. Defense compounds were found only in colonies far from pollution sources and correlated with bacteria associated with healthier environments. In contrast, pathogenic and sewage-associated bacteria dominated near the polluted site, where defenseless colonies of P. dilatata showed more disease and impaired health. Our results indicate that microbial pollution affects the capacity to modulate the microbiome through the use of infochemicals and leads to disruption of symbiosis and loss of chemical defense.

Chemical defense is a pivotal strategy for predator, pathogen, and parasite avoidance in amphibians. Although most defensive chemicals in amphibians are obtained through biosynthesis, poison frogs sequester dietary alkaloids, in some cases hydroxylating or N-methylating the ingested alkaloids to form more potent chemicals. Although more than 1200 lipophilic alkaloids have been isolated in anuran secretions, the mechanisms underlying alkaloid sequestration and biotransformation remain largely unknown. This study investigates the biochemical pathways employed by Adelphobates galactonotus to sequester and transform ingested alkaloids. Specifically, we focused on the hydroxylation of orally administered pumiliotoxin (+)-251D to form allopumiliotoxin (+)267A using ultra performance liquid chromatography - triple quadrupole mass spectrometry. We examined the distribution of these alkaloids across several organs, including the liver, skin, digestive tract, gallbladder, kidney, and fat bodies. Using a novel protocol for sample preparation via solid-phase extraction, our study elucidates the translocation patterns of these alkaloids, along with differences in their ratios. We detected pumiliotoxin (+)-251D, and its hydroxylated derivate in the liver, suggesting hepatic involvement in processing, while the skin was the primary site of storage for both alkaloids. Additionally, we report the presence of these alkaloids in the urine and fat bodies, suggesting a passive storage mechanism. This study advances our understanding of anuran chemical defense and metabolic adaptations.

Egg and early-instar morphology of Lymantria juglandis (Lepidoptera: Erebidae: Lymantriinae): Insights into its functional implications.

Amphibian skin peptides: Diversity, biological functions, and research progress.

Fusarium Head Blight (FHB) is a devastating fungal disease of small grain cereals like wheat and barley, causing substantial yield and quality losses each year worldwide. FHB is caused by Fusarium species that produce mycotoxins such as deoxynivalenol (DON) that impairs protein biosynthesis. Although defense responses in barley to Fusarium infection have been described at the transcriptional level, it remains unclear to what extent these responses are translated into functional changes at the protein and metabolite levels.In this study, we employed comprehensive transcriptomics, proteomics, and metabolomics to dissect the defense responses of barley heads during infection with Fusarium culmorum. Our integrated analyses revealed a set of significantly regulated gene-protein pairs linked to biosynthetic pathways that consistently correspond to upregulated defense-related metabolites. These include tryptophan-derived stress metabolites such as tryptamine and serotonin, as well as barley-specific hydroxycinnamylamides, including conjugates from the trypthophan metabolism, hordatines, and their biosynthetic precursors.Integrating data across multiple omics layers identifies the upregulation of aromatic amino acid derived secondary metabolism as the most consistent barley response to FHB infection across diverse barley varieties that share barley-typical type II resistance to fungal spreading in the head rachis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s44154-026-00313-5.

Cell-type-specific compartmentalization and function of the glucosinolate-myrosinase system in Arabidopsis thaliana.

Frogs produce and secrete cutaneous antimicrobial peptides (AMPs), which serve as chemical defense against microbial infections and have great biotechnological potential. AMPs are stored in intracellular vesicles in skin glands as propeptides. Before secretion, proprotein convertases (PCs) cleave propeptides into acidic spacer peptides and bioactive peptides. Identifying the correct cleavage site between the acidic spacer and bioactive peptides is a crucial step in AMP prediction. Here, we present Frog Propeptide Cleavage Site Predictor (FrogPCSP), an SVM-based predictor designed to identify propeptide cleavage sites in frog AMPs. The SVM model showed strong performance (global accuracy = 0.981, precision = 0.937, recall = 0.928, F1-score = 0.933, PR-AUC: 0.916) under grouped and stratified 10-fold cross-validation on 424 positive and 2488 negative cleavage sites. Overall, FrogPCSP demonstrated superior performance for propeptide cleavage site prediction of frog AMPs (AUC = 0.990) relative to PSSM (AUC = 0.974) and ProP (AUC = 0.905), a general-purpose reference prohormone cleavage site predictor. As proof of concept, 595 unlabeled frog AMP sequences from UniProtKB/TrEMBL were analyzed. FrogPCSP inferred 926 putative propeptide cleavage sites. Computational physicochemical profiling of the resulting peptides revealed two distinct clusters, one positively charged, with high isoelectric point and strong amphipathicity-consistent with putative bioactive peptides-and another negatively charged, with low isoelectric point and low amphipathicity values-corresponding to putative acidic spacer peptides. Thus, we believe identifying propeptide cleavage sites will assist the discovery and advance the understanding of novel frog AMPs.

Bioactive specialized metabolites (SMs) are synthesized and sequestered in specific cellular compartments or organelles as a self-defense strategy against their intrinsic toxicity. Liverwort-specific oil bodies accumulate large amounts of SMs and contribute to chemical defense; however, the molecular mechanisms underlying SM sequestration in oil bodies remain largely unknown. Therefore, in this study, we focused on MpABCG1 and MpABCG36, which are ATP-binding cassette (ABC) protein family members localized to the oil bodies of liverwort Marchantia polymorpha. Sesquiterpene (thujopsene, chamigrene, and himachalane) accumulation was reduced in the Mpabcg1 and Mpabcg36 loss-of-function mutants. Notably, levels of the bisbibenzyls, marchantins C and A, were predominantly reduced in Mpabcg1, but not in Mpabcg36. Although the Mpabcg1 mutant formed a number of oil bodies labeled with mCitrine-MpSYP12B (oil body membrane marker) comparable to that of the wild-type, the number of oil bodies stained with BODIPY 493/503, which has an affinity for lipophilic SMs, was reduced. This finding suggests that MpABCG1 and MpABCG36 mutations affect SM accumulation in the oil body but have little impact on oil body formation. Overall, our results highlight the involvement of MpABCG1 and MpABCG36 in the accumulation of SMs and/or their precursors in liverwort oil bodies.

Plants have evolved sophisticated innate immune systems to combat pathogens, starting with pattern recognition receptors that detect microbial signals to trigger PAMP-triggered immunity (PTI), countered by pathogen effectors that lead to effector-triggered immunity (ETI) via resistance proteins. Systemic defenses like salicylic acid-driven systemic acquired resistance (SAR) and jasmonic acid/ethylene-dependent induced systemic resistance (ISR) amplify protection. Specialized metabolites, such as carnosic acid, selectively activate JA/ET pathways against necrotrophs, such as Botrytis cinerea, in Arabidopsis thaliana, as shown by phenotypic and gene expression analyses (e.g., PDF1.2 upregulation). Bioinformatics tools advance this field by enabling transcriptome profiling, R-gene identification via databases such as PRGdb, and the prediction of stress-responsive pathways, offering future insights for crop improvement and chemical defense studies.