Insect Molecular Biology最新文献

{"title":"CRISPR/Cas9-mediated knockout of the abdominal-B homeotic gene in the global pest, fall armyworm (Spodoptera frugiperda).","authors":"Xiao-Guang Liu, Te Zhao, Qi-Qi Qiu, Cong-Ke Wang, Tian-Liang Li, Xiao-Long Liu, Li Wang, Qin-Qin Wang, Lin Zhou","doi":"10.1111/imb.12958","DOIUrl":"https://doi.org/10.1111/imb.12958","url":null,"abstract":"<p><p>The Homeotic complex (Hox) genes play a crucial role in determining segment identity and appendage morphology in bilaterian animals along the antero-posterior axis. Recent studies have expanded to agricultural pests such as fall armyworm (FAW), scientifically known as Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae), which significantly threatens global agricultural productivity. However, the specific role of the hox gene Sfabd-B in FAW remains unexplored. This research investigates the spatial and temporal expression patterns of Sfabd-B in various tissues at different developmental stages using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, we explored the potential function of the Sfabd-B gene located in the FAW genome using CRISPR/Cas9 technology. The larval mutant phenotypes can be classified into three subgroups as compared with wild-type individuals, that is, an excess of pedis in the posterior abdomen, deficient pedis due to segmental fusion and deviations in the posterior abdominal segments. Importantly, significant differences in mutant phenotypes between male and female individuals were also evident during the pupal and adult phases. Notably, both the decapentaplegic (dpp) and cuticular protein 12 (cp 12) genes displayed a substantial marked decrease in expression levels in the copulatory organ of male mutants and the ovipositor of female mutants compared with the wild type. These findings highlight the importance of Sfabd-B in genital tract patterning, providing a potential target for improving genetic control.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307696","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":"Foraging in the darkness: Highly selective tuning of below-ground larval olfaction to Brassicaceae volatiles in striped flea beetle.","authors":"Yong Xiao, Chunmei Lei, Xue Wang, Raufa Batool, Fei Yin, Zhengke Peng, Xiangfeng Jing, Zhenyu Li","doi":"10.1111/imb.12960","DOIUrl":"https://doi.org/10.1111/imb.12960","url":null,"abstract":"<p><p>The olfactory system of above-ground insects is among the best described perceptual architectures. However, remarkably little is known about how below-ground insects navigate in the dark for foraging. Here, we investigated host plant preferences, olfactory sensilla and characterise olfactory proteins in below-ground larvae of the striped flea beetle (SFB) Phyllotreta striolata Fabricius (Coleoptera: Chrysomelidae). Both the adults and larvae of this coleopteran pest cause serious damage to Brassicaceous crops above and below ground, respectively. To elucidate the role of olfactory system in host location of below-ground larvae, we initially demonstrated that SFB larvae distinctly favoured Brassicaceae over other plant families by two-choice behavioural bioassay. Subsequently, scanning electron microscopy of sensilla in SFB larval head showed a significant reduction in the number of olfactory sensilla in larvae compared with adults. However, essential olfactory sensilla such as sensilla basiconica are underscoring the indispensability of the larval olfactory system. We selected four larval-specific odorant binding proteins for functional validation from our previous transcriptome data. Functional studies revealed that PstrOBP23 exhibits robust binding affinity to 24 volatiles of Brassicaceae plants, including seven isothiocyanate compounds. This suggests a pivotal role of PstrOBP23 in the foraging behaviour of the larvae below the ground. Moreover, two ligands displaying strong binding capacity exhibit apparent attractive or repellent activity towards SFB larvae. Our findings provide a crucial insight into the olfactory system of below-ground larvae in SFB, highlighting the highly selective tuning of larvae specific OBP to host plant volatiles. These results offer potential avenues for developing effective pest control strategies against SFB.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142286194","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":"Alternative double strand break repair pathways shape the evolution of high recombination in the honey bee, Apis mellifera.","authors":"Bertrand Fouks,Katelyn J Miller,Caitlin Ross,Corbin Jones,Olav Rueppell","doi":"10.1111/imb.12961","DOIUrl":"https://doi.org/10.1111/imb.12961","url":null,"abstract":"Social insects, particularly honey bees, have exceptionally high genomic frequencies of genetic recombination. This phenomenon and underlying mechanisms are poorly understood. To characterise the patterns of crossovers and gene conversion in the honey bee genome, a recombination map of 187 honey bee brothers was generated by whole-genome resequencing. Recombination events were heterogeneously distributed without many true hotspots. The tract lengths between phase shifts were bimodally distributed, indicating distinct crossover and gene conversion events. While crossovers predominantly occurred in G/C-rich regions and seemed to cause G/C enrichment, the gene conversions were found predominantly in A/T-rich regions. The nucleotide composition of sequences involved in gene conversions that were associated with or distant from crossovers corresponded to the differences between crossovers and gene conversions. These combined results suggest two types of DNA double-strand break repair during honey bee meiosis: non-canonical homologous recombination, leading to gene conversion and A/T enrichment of the genome, and the canonical homologous recombination based on completed double Holliday Junctions, which can result in gene conversion or crossover and is associated with G/C bias. This G/C bias may be selected for to balance the A/T-rich base composition of eusocial hymenopteran genomes. The lack of evidence for a preference of the canonical homologous recombination for double-strand break repair suggests that the high genomic recombination rate of honey bees is mainly the consequence of a high rate of double-strand breaks, which could in turn result from the life history of honey bees and their A/T-rich genome.","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":"12 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142255854","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":"Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.","authors":"Jacob N Dayton,Tammy T Tran,Elisa Saint-Denis,Erik B Dopman","doi":"10.1111/imb.12959","DOIUrl":"https://doi.org/10.1111/imb.12959","url":null,"abstract":"The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":"2 1","pages":""},"PeriodicalIF":2.6,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268916","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":"Changes in bumblebee queen gut microbiotas during and after overwintering diapause.","authors":"Michelle Z Hotchkiss, Jessica R K Forrest, Alexandre J Poulain","doi":"10.1111/imb.12957","DOIUrl":"https://doi.org/10.1111/imb.12957","url":null,"abstract":"<p><p>Bumblebees are key pollinators with gut microbiotas that support host health. After bumblebee queens undergo winter diapause, which occurs before spring colony establishment, their gut microbiotas are disturbed, but little is known about community dynamics during diapause itself. Queen gut microbiotas also help seed worker microbiotas, so it is important that they recover post-diapause to a typical community structure, a process that may be impeded by pesticide exposure. We examined how bumblebee queen gut microbiota community structure and metabolic potential shift during and after winter diapause, and whether post-diapause recovery is affected by pesticide exposure. To do so, we placed commercial Bombus impatiens queens into diapause, euthanizing them at 0, 2 and 4 months of diapause. Additionally, we allowed some queens to recover from diapause for 1 week before euthanasia, exposing half to the common herbicide glyphosate. Using whole-community, shotgun metagenomic sequencing, we found that core bee gut phylotypes dominated queen gut microbiotas before, during and after diapause, but that two phylotypes, Schmidhempelia and Snodgrassella, ceased to be detected during late diapause and recovery. Despite fluctuations in taxonomic community structure, metabolic potential remained constant through diapause and recovery. Also, glyphosate exposure did not affect post-diapause microbiota recovery. However, metagenomic assembly quality and our ability to detect microbial taxa and metabolic pathways declined alongside microbial abundance, which was substantially reduced during diapause. Our study offers new insights into how bumblebee queen gut microbiotas change taxonomically and functionally during a key life stage and provides guidance for future microbiota studies in diapausing bumblebees.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142035757","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":"A sex-specific homologue of waprin is essential for embryonic development in the red flour beetle, Tribolium castaneum.","authors":"Chhavi Choudhary, Divyanshu Kishore, Keshav Kumar Meghwanshi, Vivek Verma, Jayendra Nath Shukla","doi":"10.1111/imb.12956","DOIUrl":"https://doi.org/10.1111/imb.12956","url":null,"abstract":"<p><p>Waprin, a WAP (Whey acidic protein) domain-containing extracellular secretory protein, is widely known for its antibacterial properties. In this study, a waprin homologue (Tc_wap^F) expressing in a female-specific manner was identified in Tribolium castaneum, through the analysis of sex-specific transcriptomes. Developmental- and tissue-specific profiling revealed the widespread expression of Tc_wap^F in adult female tissues, particularly in the ovary, gut and fatbody. This female-specific expression of Tc_wap^F is not regulated by the classical sex-determination cascade of T. castaneum, as we fail to get any attenuation in Tc_wap^F transcript levels in Tcdsx and Tctra (key players of sex determination cascade of T. castaneum) knockdown females. RNA interference-mediated knockdown of Tc_wap^F in females led to the non-hatching of eggs laid by these females, suggesting the crucial role of Tc_wap^F in the embryonic development in T. castaneum. This is the first report on the identification of a sex-specific waprin homologue in an insect and its involvement in embryonic development. Future investigations on the functional conservation of insect waprins and their mechanistic role in embryonic development can be exploited for improving pest management strategies.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142017313","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":"Minibrain plays a role in the adult brain development of honeybee (Apis mellifera) workers.","authors":"Juliana Ramos Martins, Izabella Cristina Silva, Talita Sarah Mazzoni, Gabriela Helena de Barrios, Flávia Cristina de Paula Freitas, Angel Roberto Barchuk","doi":"10.1111/imb.12955","DOIUrl":"https://doi.org/10.1111/imb.12955","url":null,"abstract":"<p><p>The brain of adult honeybee (Apis mellifera) workers is larger than that of queens, facilitating behavioural differentiation between the castes. This brain diphenism develops during the pharate-adult stage and is driven by a caste-specific gene expression cascade in response to unique hormonal milieus. Previous molecular screening identified minibrain (mnb; DYRK1A) as a potential regulator in this process. Here, we used RNAi approach to reduce mnb transcript levels and test its role on brain diphenism development in honeybees. White-eyed unpigmented cuticle worker pupae were injected with dsRNA for mnb (Mnb-i) or gfp, and their phenotypes were assessed two and 8 days later using classic histological and transcriptomic analyses. After 2 days of the injections, Mnb-i bees showed 98% of downregulation of mnb transcripts. After 8 days, the brain of Mnb-i bees showed reduction in total volume and in the volume of the mushroom bodies (MB), antennal, and optic lobes. Additionally, signs of apoptosis were observed in the Kenyon cells region of the MB, and the cohesion of the brain tissues was affected. Our transcriptomic analyses revealed that 226 genes were affected by the knockdown of mnb transcripts, most of which allowing axonal fasciculation. These results suggest the evolutionary conserved mnb gene has been co-opted for promoting hormone-mediated developmental brain morphological plasticity generating caste diphenism in honeybees.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142017314","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":"Bombyx mori nucleopolyhedrovirus LEF-2 disrupts the cell cycle in the G2/M phase by triggering a host cell DNA damage response.","authors":"Jie Wang, Miao Xiao, Zhigang Hu, Yu Lin, Kejie Li, Peng Chen, Cheng Lu, Zhanqi Dong, Minhui Pan","doi":"10.1111/imb.12951","DOIUrl":"https://doi.org/10.1111/imb.12951","url":null,"abstract":"<p><p>It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141987893","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":"Spraying dsRNA with chitosan formulation improves control of the western flower thrips, Frankliniella occidentalis, in a greenhouse.","authors":"Falguni Khan, Gahyeon Jin, Yonggyun Kim","doi":"10.1111/imb.12954","DOIUrl":"https://doi.org/10.1111/imb.12954","url":null,"abstract":"<p><p>The western flower thrips, Frankliniella occidentalis, is a serious pest causing both direct feeding damage and indirect harm by transmitting the tomato spotted wilt virus. A spraying double-stranded RNA (dsRNA) targeted at the vacuolar-type ATPase (vATPase) gene was developed and demonstrated high insecticidal activity in the laboratory but less effective in field applications. To improve control efficacy under field conditions, three strategies were explored in this study. First, to identify a more efficient RNA interference (RNAi) target, dsRNA specific to the Snf7 gene was tested alongside dsRNA targeting vATPase, and both were found to be similarly effective in controlling the thrips. Second, to elucidate the factors contributing to dsRNA resistance, dsRNA-degrading enzymes were annotated and their physiological roles in diminishing RNAi efficacy were investigated. Third, to suppress the dsRNA degradation from the dsRNase activities and protect it in field conditions, the dsRNA was encapsulated with chitosan. This formulation enhanced the dsRNA's resistance to environmental stressors such as ultraviolet light and the digestive enzymes in the thrips' gut. Additionally, the chitosan formulation specifically increased the RNAi efficacy, likely by facilitating more efficient entry into the target cells, thus bolstering the insecticidal activity of the dsRNA. The formulated dsRNA was applied on F. occidentalis infesting the hot peppers in a greenhouse at a concentration of 500 ppm, demonstrating an 82.4% control efficacy compared with 59.2% control efficacy observed with the application of naked dsRNA. This study further demonstrated an enhancement in the spectrum of control by combining dsRNAs specific to three distinct thrips species, while the mixture showed no adverse effects on non-target insects, such as the lepidopteran Spodoptera exigua. Collectively, these findings reveal that the chitosan formulation of dsRNA not only improves control efficacy under field conditions but also broadens the control spectrum against three different thrips pests.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141971076","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":"Expanding the transgene expression toolbox of the malaria vector Anopheles stephensi.","authors":"Joshua Southworth, Estela Gonzalez, Katherine Nevard, Mireia Larrosa-Godall, Luke Alphey, Michelle A E Anderson","doi":"10.1111/imb.12953","DOIUrl":"https://doi.org/10.1111/imb.12953","url":null,"abstract":"<p><p>Anopheles stephensi Liston, 1901 (Diptera: culicidae) is a competent vector of Plasmodium falciparum (Haemosporida: plasmodiidae) malaria, and its expansion in the African continent is of concern due to its viability in urban settings and resistance to insecticides. To enhance its genetic tractability, we determined the utility of a ~2 kb An. stephensi lipophorin (lp) promoter fragment in driving transgene expression. Lipophorin genes are involved in lipid transport in insects, and an orthologous promoter in An. gambiae (AGAP001826) was previously demonstrated to successfully express a transgene. In the present study, we qualitatively characterised the expression of a ZsYellow fluorescent marker protein, expressed by An. stephensi lp promoter fragment. Our study indicated that the lp promoter fragment was effective, generating a distinct expression pattern in comparison to the commonly utilised 3xP3 promoter. The lp:ZsYellow fluorescence was largely visible in early instar larvae and appeared more intense in later instar larvae, pupae and adults, becoming especially conspicuous in adult females after a blood meal. Different isolines showed some variation in expression pattern and intensity. Aside from general transgene expression, as the lp promoter produces a suitable fluorescent protein marker expression pattern, it may facilitate genotypic screening and aid the development of more complex genetic biocontrol systems, such as multi-component gene drives. This study represents an expansion of the An. stephensi genetic toolbox, an important endeavour to increase the speed of An. stephensi research and reach public health milestones in combating malaria.</p>","PeriodicalId":13526,"journal":{"name":"Insect Molecular Biology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141916556","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}