Insect Biochemistry and Molecular Biology最新文献

{"title":"Corrigendum to \"The genome sequence of the Neotropical brown stink bug, Euschistus heros provides insights into population structure, demographic history and signatures of adaptation\" [Insect Biochem. Mol. Biol. 152, (2023), 103890].","authors":"Kumar Saurabh Singh, Erick M G Cordeiro, Benjamin J Hunt, Aniruddha A Pandit, Patricia L Soares, Alberto S Correa, Christoph T Zimmer, Maria I Zucchi, Carlos Batista, Julian A T Dow, Shireen-Anne Davies, Fernando Luís Cônsoli, Celso Omoto, Chris Bass","doi":"10.1016/j.ibmb.2026.104689","DOIUrl":"https://doi.org/10.1016/j.ibmb.2026.104689","url":null,"abstract":"","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":" ","pages":"104689"},"PeriodicalIF":4.3,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149340142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vapors of juvenile hormone accelerate progression through the pupal stage and block adult development.","authors":"Mária Gondová, Matěj Miláček, Jakub Žák, Tibor Füzik, Kateřina Kolářová, Pavel Plevka, Marek Jindra, Antonín Přidal","doi":"10.1016/j.ibmb.2026.104690","DOIUrl":"https://doi.org/10.1016/j.ibmb.2026.104690","url":null,"abstract":"<p><p>Complete metamorphosis of insects is regulated by mutually interacting juvenile hormone (JH) and ecdysteroid signals. The holometabolous pupa is a stage at which a natural absence of JH permits adult morphogenesis. Administration of exogenous JH or synthetic JH mimics at this stage disrupts development, but details of the ectopic JH action are not well understood. Here, using time-resolved imaging in the red flour beetle (Tribolium castaneum) and the honey bee (Apis mellifera), we show that treatment with the native hormone JH III not only blocks the pupa-adult transition, but also accelerates the appearance of morphological traits and shortens the total duration of the pupal stage. As assessed in T. castaneum, these effects were accompanied by altered expression of genes regulating metamorphosis and by reduced ecdysteroid levels, thus likely causing heterochronic developmental shifts resulting from endocrine imbalance. Pupae of both species were equally affected by direct application of JH III on the pupal cuticle surface and by contactless exposure to JH III vapors. Like the topical application, the vapor-phase hormone activated the established JH signaling pathway hallmarked by transcriptional induction of the anti-metamorphic gene Krüppel-homolog 1 (Kr-h1). This gaseous JH action is relevant to experimental settings and to applications in insect pest management, apiculture, and insect mass rearing. Our findings broaden understanding of JH impact on metamorphosis, address the mechanism of volatile JH effect, and suggest that JH might play a role as an airborne endocrine signal acting at short distances.</p>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":" ","pages":"104690"},"PeriodicalIF":4.3,"publicationDate":"2026-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149340143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PGRP-S2 contributes to Toll-1-dependent antiviral immunity and apoptosis-associated signaling during IAPV infection in honey bees.","authors":"Jie Zhuang, Yanchun Deng, Man Liu, Dahe Yang, Hongmei Jin, Ghulam Jaffar, Sa Yang, Chunying Yuan, Xiaoli Qin, Xiai Yang, Hongxia Zhao, Mingxiao Ma, Chunsheng Hou","doi":"10.1016/j.ibmb.2026.104688","DOIUrl":"https://doi.org/10.1016/j.ibmb.2026.104688","url":null,"abstract":"<p><p>The Toll pathway mediates antimicrobial peptide (AMP) synthesis and participates in antiviral innate immunity in insects. However, how Toll-dependent antiviral responses are regulated during viral infection and how AMPs mediate antiviral defense remain incompletely understood. Here, we found that the Toll pathway constitutes an important part of the transcriptional response to Israeli acute paralysis virus (IAPV) infection in honey bees. PGRP-S2 was markedly up-regulated upon infection and exhibited physical associations with the IAPV capsid protein VP3 and with Toll-1, as validated by complementary co-immunoprecipitation, pull-down, and immunofluorescence analyses. Silencing PGRP-S2 substantially elevated viral titers and diminished the expression of Toll-dependent AMPs, including defensin 1. Functional assays indicated that defensin 1 was associated with apoptosis-related responses and suppressing viral replication. Knockdown of defensin 1 correlated with reduced apoptosis-associated signaling and enhanced viral titers, whereas overexpression of defensin 1 in Sf9 cells was associated with cell death. Collectively, our findings support a contribution of PGRP-S2 to the Toll-1-dependent antiviral response and link defensin 1 to apoptosis-related responses during IAPV infection, providing new insights into antiviral immunity in honey bees.</p>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":" ","pages":"104688"},"PeriodicalIF":4.3,"publicationDate":"2026-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149316752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “Pipiserpin orchestrates mosquito reproduction through dual control of vitellogenin integrity and 20-hydroxyecdysone-directed vitellogenesis” [Insect Biochem. Molec. Biol. 189 (2026) 104514]","authors":"Yinghui Zhou ,&nbsp;Yunxuan Chen ,&nbsp;Chanuka Wijewardana ,&nbsp;Chenxuan Jiang ,&nbsp;Yuhan Guo ,&nbsp;Donghui Zhang ,&nbsp;Min Hou ,&nbsp;Zhipeng Xu ,&nbsp;Lu Chen ,&nbsp;MinJun Ji ,&nbsp;Lin Chen","doi":"10.1016/j.ibmb.2026.104517","DOIUrl":"10.1016/j.ibmb.2026.104517","url":null,"abstract":"","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104517"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146193730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BdorOR49a and BdorOR63a are responsible for male pheromones perception in the oriental fruit fly","authors":"Fu Cao ,&nbsp;Hongfei Li ,&nbsp;Bao Dong ,&nbsp;Yuanyuan Peng ,&nbsp;Li Xu ,&nbsp;Shifan Li ,&nbsp;Jinjun Wang ,&nbsp;Hongbo Jiang","doi":"10.1016/j.ibmb.2026.104503","DOIUrl":"10.1016/j.ibmb.2026.104503","url":null,"abstract":"<div><div>The oriental fruit fly, <em>Bactrocera dorsalis</em> is a serious fruit pest. Trimethylpyrazine (TMP) and tetramethylpyrazine (TTMP) were characterized as its male released sex pheromone, which showed strong attraction to sexually mature females. So far, the molecular basis of the perception of TMP and TTMP by the females has remained unknown. In this study, using <em>BdorIR8a</em>^{<em>−/−</em>} and <em>BdorOrco</em>^{<em>−/−</em>} mutants, we demonstrated that the perception of TMP and TTMP is mediated by odorant receptors (ORs). Furthermore, we generated transgenic <em>Drosophila</em> expressing two odorant receptors (<em>BdorOR49a</em> and <em>BdorOR63a</em> previously showed <em>in vitro</em> binding ability to TMP and TTMP, respectively) in T1 neurons. Subsequent single sensillum recordings (SSR) of the transgenic fly demonstrated that <em>BdorOR49a</em> and <em>BdorOR63a</em> are tuned to TMP and TTMP, respectively. Furthermore, knockout of <em>BdorOR49a</em> and knockdown of <em>BdorOR63a in vivo</em> reduced the olfactory sensitivity of the females to TMP and TTMP. Our results indicated that <em>BdorOR49a</em> and <em>BdorOR63a</em> are responsible for the perception of the male released sex pheromones in <em>B</em>. <em>dorsalis</em>.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104503"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146096881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signaling pathway underlying autocrine activation of ecdysteroidogenesis in prothoracic glands of Bombyx mori","authors":"Shi-Hong Gu","doi":"10.1016/j.ibmb.2026.104515","DOIUrl":"10.1016/j.ibmb.2026.104515","url":null,"abstract":"<div><div>Autocrine activation of ecdysteroidogenesis in the prothoracic glands (PGs) of the silkworm, <em>Bombyx mori</em>, was previously demonstrated. In the present study, the signaling cascade involved in this autocrine activation was further investigated. I found that phosphorylation of the translational repressor 4E-binding protein (4E-BP) increased in a time-dependent manner when PGs were incubated in a small volume (10 μl) of medium, compared to those incubated in a larger volume (100 μl). Higher levels of 4E-BP phosphorylation were also observed in PGs incubated either in PG-conditioned medium or under group incubation conditions. Treatment with LY294002 and rapamycin reduced 4E-BP phosphorylation, indicating the involvement of the PI3K/TOR pathway. I further investigated the downstream target genes involved in autocrine activation. Results showed that the expression levels of several <em>sugar transporter</em> (<em>St</em>) and <em>trehalase</em> (<em>Treh</em>) genes were upregulated in PGs incubated in a small volume (10 μl) of medium. Treatment with LY294002 and rapamycin suppressed the autocrine activation of <em>St1</em> and <em>Treh1</em> expressions, suggesting that their regulation is mediated via the PI3K/TOR pathway. Time-dependent autocrine activation of Treh enzyme activity was observed, and this effect was blocked by pretreatment with validamycin A, a specific Treh inhibitor, suggesting that the autocrine factor directly enhances Treh activity. Treatment with either validamycin A or the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) suppressed autocrine activation of ecdysteroidogenesis, clearly indicating that both Treh activity and glycolysis are involved. To investigate potential upstream ligands, I examined the gene expressions of <em>bombyxins</em> and epidermal growth factor (Egf) signaling components. Results showed that only <em>bombyxin-Z1</em> expression was upregulated in a time-dependent manner under an autocrine condition, while other genes remained unchanged. Using a specifically generated anti-bombyxin-Z1 antibody, the presence of bombyxin-Z1 protein was confirmed. To my knowledge, this is the first study to elucidate the signaling cascade involved in autocrine activation of ecdysteroidogenesis in an insect system.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104515"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146136993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pipiserpin orchestrates mosquito reproduction through dual control of vitellogenin integrity and 20-hydroxyecdysone-directed vitellogenesis","authors":"Yinghui Zhou ,&nbsp;Yunxuan Chen ,&nbsp;Chanuka Wijewardana ,&nbsp;Chenxuan Jiang ,&nbsp;Yuhan Guo ,&nbsp;Donghui Zhang ,&nbsp;Min Hou ,&nbsp;Zhipeng Xu ,&nbsp;Lu Chen ,&nbsp;MinJun Ji ,&nbsp;Lin Chen","doi":"10.1016/j.ibmb.2026.104514","DOIUrl":"10.1016/j.ibmb.2026.104514","url":null,"abstract":"<div><div>Mosquito-borne diseases persist as a critical global health burden, driving the demand for novel vector control approaches. Here, we identify and characterize Pipiserpin, a female-specific serine protease inhibitor (serpin) in <em>Culex pipiens pallens</em>, and elucidate its role in regulating reproductive fitness. Pipiserpin exhibits tissue-specific expression in the fat body and ovary, peaking post-blood meal, and features a conserved serpin fold with a reactive central loop critical for protease inhibition. RNA interference-mediated knockdown of Pipiserpin severely impaired ovarian follicular development, reducing follicle size by 7.73% and egg production by 68.10%. Mechanistically, Pipiserpin depletion destabilized vitellogenin (Vg)—a key yolk precursor—by 62.79% in the fat body and 73.01% in the ovary, despite unchanged Vg transcription. This phenotype was attributed to unchecked trypsin-mediated Vg degradation, as demonstrated by recombinant Pipiserpin's dose-dependent inhibition of trypsin activity. Crucially, exogenous 20-hydroxyecdysone (20E) supplementation restored Vg protein levels, follicular maturation and fecundity, revealing a compensatory relationship wherein the 20E-Vg hormonal pathway can functionally overcome reproductive defects caused by Pipiserpin depletion. Global transcriptomic profiling of Pipiserpin-depleted mosquitoes identified 221 differentially expressed genes, including upregulated trypsin-like proteases and metabolic pathway components, indicative of disrupted proteolytic balance and compensatory metabolic remodeling. Our findings redefine serpin functionality in arthropods, highlighting Pipiserpin's dual role in post-translational Vg stabilization and crosstalk with developmental signaling. This work positions Pipiserpin as a promising target for RNAi or small-molecule interventions to disrupt mosquito reproduction, offering a sustainable strategy for combating vector-borne diseases by precisely targeting reproduction.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104514"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146117235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Knockout of nAChR subunits in spider mites and their phytoseiid predators confers spinosyn cross-resistance and reveals a conserved mode of action in mites","authors":"Antonio Mocchetti ,&nbsp;Pieter Steelant ,&nbsp;Mahboubeh Hosseinkhani ,&nbsp;Sander De Rouck ,&nbsp;Jahangir Khajehali ,&nbsp;Thomas Van Leeuwen","doi":"10.1016/j.ibmb.2026.104498","DOIUrl":"10.1016/j.ibmb.2026.104498","url":null,"abstract":"<div><div>Spinosyns are allosteric modulators of nicotinic acetylcholine receptors (nAChRs) which in insects specifically target subunit α6. However, their mode of action in mites and compatibility with phytoseiid predators remain unclear. We combined phylogenetics with CRISPR/Cas-based reverse genetics to test whether α6-like subunits mediate spinosyn toxicity in mites and to assess prospects for resistance breeding in phytoseiids. The phylogenetic analysis identified seven α and three β subunits in multiple phytoseiids and in <em>Tetranychus urticae</em>. A single phytoseiid subunit clustered within the insect α6/α7 clade, whereas <em>T. urticae</em> possessed three (Tuα5/α6/α7) without strict one-to-one insect orthology. Using SYNCAS maternal delivery of CRISPR RNPs, we disrupted the putative α6 ortholog in <em>Amblyseius swirskii</em> (Asα6) and each of the three α6/α7-clade genes in <em>T. urticae</em>. In <em>A. swirskii</em>, all survivors of a discriminating spinosad dose carried Asα6 indels, and three independently edited lines exhibited insensitivity to both spinosad and spinetoram (no significant mortality at 10 000 mg a.i./L), whereas the wild type showed LC₅₀ = 163 mg/L (spinosad) and 54 mg/L (spinetoram). In <em>T. urticae</em>, Tuα6 knockouts conferred high cross-resistance to both compounds, while Tuα5 knockouts slightly increased susceptibility and Tuα7 knockouts produced modest resistance. Our data demonstrate that α6-mediated spinosyn action is conserved in mites, with α6 loss conferring strong cross-resistance in a key phytoseiid predator and in a model tetranychid. These findings enable marker-assisted editing/selection of spinosyn-resistant phytoseiid strains to improve pesticide–biocontrol compatibility and establish α6 as a practical universal marker gene for genome editing in acarine systems.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104498"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146040050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of tomato leaf miner secretory proteins and their roles in influencing plant defenses","authors":"Yumei Dong ,&nbsp;Guolan Wu ,&nbsp;Qiuyun Zhang ,&nbsp;Zhili Zhao ,&nbsp;Yunhua Zhang ,&nbsp;Qian Li ,&nbsp;Chuanlin Yin ,&nbsp;Pengjun Zhang","doi":"10.1016/j.ibmb.2026.104500","DOIUrl":"10.1016/j.ibmb.2026.104500","url":null,"abstract":"<div><div>The tomato leaf miner (<em>Tuta absoluta</em>) is a globally destructive pest that cause extensive damage to tomato crops by chewing mouthparts, leading to severe necrosis, fruit abortion, and substantial yield losses. To date, the elicitors/effectors of <em>T. absoluta</em> have not been characterized. In this study, we combined proteomic profiling of <em>T. absoluta</em>-infested tomato leaves with transcriptomic analysis of salivary glands to identify candidate molecules involved in herbivory-driven plant responses. Bioinformatics analyses predicted 40 candidate elicitors and effectors, which were subsequently assessed through transient expression assays in <em>Nicotiana benthamiana</em>. The results demonstrated that the candidate number 33 (<em>T</em>. <em>absoluta</em> 33, Ta33) induced cell death in both the intracellular space and the apoplast, while Ta21 triggered a strong apoplastic reactive oxygen species (ROS) burst. Conversely, Ta38 effectively suppressed INF1-induced cell death. Quantitative real-time PCR analysis further showed that these genes were highly expressed during the feeding stage, supporting their involvement in plant–insect molecular dialogue. This study systematically identified and characterized elicitors and effectors of <em>T. absoluta</em>, providing a foundational framework for elucidating its herbivory mechanisms and developing targeted management strategies.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104500"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146057322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rethinking insecticide toxicology for the 21st century","authors":"Lautaro Gandara ,&nbsp;Felipe Martelli ,&nbsp;Thomas Ravenscroft ,&nbsp;Justin Crocker ,&nbsp;Philip Batterham","doi":"10.1016/j.ibmb.2026.104502","DOIUrl":"10.1016/j.ibmb.2026.104502","url":null,"abstract":"<div><div>Insecticides remain indispensable for crop protection and food security, yet their widespread use may contribute to the global decline of beneficial insect populations. Efforts to mitigate these impacts are hampered by a fragmented understanding of how insects metabolise insecticides and how sublethal exposures affect physiology, behaviour, and fitness. Here, we synthesise current understanding of metabolic detoxification and highlight critical gaps: the tissue- and time-dependent dynamics of insecticide entry and processing, the triggers and architecture of xenobiotic transcriptional responses, the role of rapid non-transcriptional regulation, and the population-level consequences of sublethal effects. We also outline emerging experimental strategies for addressing these questions and propose a next-generation research pipeline centred on multi-endpoint phenomics across life stages and sentinel species, integrated with AI-driven predictive toxicology, as a framework for identifying safer chemicals. We propose an integrated framework unifying molecular, physiological, and ecological responses to sublethal exposure to guide the design of insecticides that maintain effective pest control while safeguarding insect biodiversity and the ecosystems it underpins.</div></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":"189 ","pages":"Article 104502"},"PeriodicalIF":3.7,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146096655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}