A Preliminary Toxicology Study on Eco-friendly Control Target of Spodoptera frugiperda.

Pyrethroid and indoxacarb are commonly used pesticides to control the fall armyworm (Spodoptera frugiperda) in the crops. There are a series of consequences caused by the use of pyrethroid and indoxacarb pesticides under emergency control, such as pest resistance development, contamination of soil, water, and farm products. This study analyzed the structure and physiological function of the sodium channel in S. frugiperda, which is an important step to elaborate the resistance mechanism of S. frugiperda to indoxacarb and pyrethroid pesticides. According to genetic analysis, the cloned cDNA sequences of sodium channel in S. frugiperda (SfNav) showed the shortest genetic distance with that of the sodium channel in Helicoverpa armigera. Under the induction of three pesticides, the expression of SfNav decreased in the first 12 h and then increased after 24 h. It was concluded that SfNav had a typical structure of the sodium channel of insects and its down-regulated expression can decrease the combination of S. frugiperda with pyrethroid and indoxacarb pesticides. The up-regulated expression of SfNav was conducive to the enhancement of the pesticide resistance.

Genomic evaluations of Wolbachia and mtDNA in the population of coconut hispine beetle, Brontispa longissima (Coleoptera: Chrysomelidae).

Wolbachia pipientis is a diverse, ubiquitous and most prevalent intracellular bacterial group of alpha-Proteobacteria that is concerned with many biological processes in arthropods. The coconut hispine beetle (CHB), Brontispa longissima (Gestro) is an economically important pest of palm cultivation worldwide. In the present study, we comprehensively surveyed the Wolbachia-infection prevalence and mitochondrial DNA (mtDNA) polymorphism in CHB from five different geographical locations, including China's Mainland and Taiwan, Vietnam, Thailand, Malaysia and Indonesia. A total of 540 sequences were screened in this study through three different genes, i.e., cytochrome oxidase subunit I (COI), Wolbachia outer surface protein (wsp) and multilocus sequencing type (MLST) genes. The COI genetic divergence ranges from 0.08% to 0.67%, and likewise, a significant genetic diversity (π = 0.00082; P = 0.049) was noted within and between all analyzed samples. In the meantime, ten different haplotypes (H) were characterized (haplotype diversity = 0.4379) from 21 different locations, and among them, H6 (46 individuals) have shown a maximum number of population clusters than others. Subsequently, Wolbachia-prevalence results indicated that all tested specimens of CHB were found positive (100%), which suggested that CHB was naturally infected with Wolbachia. Wolbachia sequence results (wsp gene) revealed a high level of nucleotide diversity (π = 0.00047) under Tajima's D test (P = 0.049). Meanwhile, the same trend of nucleotide diversity (π = 0.00041) was observed in Wolbachia concatenated MLST locus. Furthermore, phylogenetic analysis (wsp and concatenated MLST genes) revealed that all collected samples of CHB attributed to same Wolbachia B-supergroup. Our results strongly suggest that Wolbachia bacteria and mtDNA were highly concordant with each other and Wolbachia can affect the genetic structure and diversity within the CHB populations.

Population Susceptibility to Insecticides and the Development of Resistance in Bactrocera cucurbitae (Diptera: Tephritidae).

Excessive insecticide applications are commonly used to manage Bactrocera cucurbitae Coquillett in China. Resistance status, resistance development trends, and patterns of cross-resistance to insecticides in B. cucurbitae were investigated. Among 21 populations from Hainan Island, two populations expressed high resistance to beta-cypermethrin; seven, eight, and ten populations expressed intermediate resistance to spinosad, avermectin, and beta-cypermethrin, respectively; four, six, one, five, and four populations expressed low resistance to spinosad, avermectin, trichlorfon, beta-cypermethrin, and fipronil, respectively; and the remaining populations exhibited either minor resistance or remained susceptible. Analysis of the development of resistance showed that resistance levels to spinosad and avermectin were readily developed at 40.68- and 18.42-fold, respectively, and a spinosad-resistant strain also showed relative positive cross-resistance to beta-cypermethrin and avermectin, but relative negative cross-resistance to trichlorfon and fipronil. These data represent the most extensive survey of insecticide resistance conducted in B. cucurbitae to date, and the level of insecticide resistance in populations should be considered when designing control measures and pest management strategies.

Prionic Acid: An Effective Sex Attractant for an Important Pest of Sugarcane, Dorysthenes granulosus (Coleoptera: Cerambycidae: Prioninae).

Male Dorysthenes granulosus (Thomson, 1860) (Coleoptera: Cerambycidae: Prioninae) were caught in traps baited with racemic 3,5-dimethyldodecanoic acid (prionic acid) during field screening trials in China that tested known cerambycid pheromones. This species is an important pest of sugarcane (Saccharum officinarum L.). In follow-up dose-response trials, plastic sachets loaded with 1 or 0.1 mg of prionic acid were equally attractive to male beetles, whereas lower doses were no better than controls. Two commercial prionic acid lures also were attractive, suggesting that traps baited with prionic acid can be rapidly incorporated into integrated pest management programs targeting this major pest. It is likely that this compound is a major component of the female-produced sex pheromone of D. granulosus because this species is in the same subfamily as Prionus californicus Motschulsky, 1845, the species from which prionic acid was originally identified.

Identification of putative odorant binding protein genes in Asecodes hispinarum, a parasitoid of coconut leaf beetle (Brontispa longissima) by antennal RNA-Seq analysis.

Asecodes hispinarum (Hymenoptera: Eulophidae) is an endoparasitoid and an efficient biological control agent which attacks larvae of Brontispa longissima, a serious insect pest of Palmae plants in China. Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. No previous study has reported on OBPs in A. hispinarum. In this study, we conducted the large-scale identification of OBP genes from the antennae of A. hispinarum by using transcriptome sequencing. Approximately 28.4 million total raw reads and about 27.3 million total clean reads were obtained, and then 46,363 unigenes were assembled. Of these unigenes, a total of 21,263 can be annotated in the NCBI non-redundant database. Among the annotated unigenes, 16,623 of them can be assigned to GO (Gene Ontology). Furthermore, we identified 8 putative OBP genes, and a phylogenetic tree analysis was performed to characterize the 8 OBP genes. In addition, the expression of the 8 OBP genes in different A. hispinarum body tissues was analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). The results indicated that the 8 OBP genes were expressed accordingly to sexes and tissues, but all highly expressed in antennae. The finding of this study will lay the foundation for unraveling molecular mechanisms of A. hispinarum chemoperception.

Effect of temperatures and cold storage on performance of Tetrastichus brontispae (Hymenoptera: Eulophidae), a parasitoid of Brontispa longissima (Coleptera: Chrysomelidae).

Laboratory studies were conducted to determine the effect of temperature and cold storage on the performance of Tetrastichus brontispae (Ferriere) (Hymenoptera: Eulophidae), one of the major endoparasitoids against coconut hispine beetle, Brontispa longissima (Gestro) (Coleptera: Chrysomelidae). The results revealed that T. brontispae could successfully parasitize host pupae under all seven tested temperatures, but no adult emergence was observed at 32°C. It was also revealed that temperatures between 24 and 26°C appeared to be the optimum temperatures for parasitism, as these temperatures resulted in the most parasitized pupae and a significantly higher emergence rate and progeny production. These measurements significantly declined at 20, 30, and 32°C. This study confirmed developmental periods of parasitoid progeny decreased as the temperature increased, and sex ratio of this female-biased parasitoid was not affected by rearing temperatures. More importantly, this study indicated that cold storage of parasitized pupae could extend up to 30 d at 10°C, and a longer storage period had a significant adverse effect on mean adult emergence and parasitism performance. Ten days might be the optimum cold-storage period at 10°C, as parasitism performance, emergence rate, and progeny production at this storage period were similar to the control of 26°C. Furthermore, the developmental period, emergence rate, and sex ratio of progeny that emerged from cold-stored parasitized pupae were not influenced by storage periods, whereas parasitism performance of progeny decreased as storage period increased. This study suggests that about 24-26°C would be the optimal temperature for mass production and release of T. brontispae for biological control of B. longissima. These results also provide novel findings that a period of 10 d at 10°C may be more suitable and acceptable for ideal cold storage of parasitized pupae of T. brontispae.

Exploring the hormetic effects of radiation on the life table parameters of Spodoptera frugiperda.

BACKGROUND: Spodoptera frugiperda, a global agricultural pest, can be effectively controlled through the sterile insect technique. However, exposure to low-dose radiation below the sterilization threshold may induce hormetic effects. Here, the biphasic aspects of the fertile progeny population of S. frugiperda were analyzed using an age-stage, two-sex life table after dosing male and female pupae with 10-350 Gy gamma radiation. RESULTS: The parental sterilizing dose for 6-day-old female and male pupae was 200 and 350 Gy, respectively. The total longevity, pre-adult survival rate, net reproduction rate, and intrinsic growth rate of the offspring population increased with decreasing radiation doses from 250 to 10 Gy. Offspring population of parents treated with low doses of 10-100 Gy showed better life table parameters compared to non-irradiated controls. Females and males fecundity irradiated with 10, 50, and 100 Gy and 10 Gy, respectively, exceeded controls, producing 2339.4, 2726.4, 2311, and 2369 eggs, as opposed to 1802.9 eggs produced by the controls. Males irradiated with 10 Gy displayed the highest intrinsic rates of increase and net reproduction rate, at 0.1709 and 682.3, respectively. Projections from the survival rate and fecundity indicated that female and male S. frugiperda populations after 10 Gy irradiation may grow considerably faster than the controls. CONCLUSION: This study explores the hormetic effects of low-dose radiation on S. frugiperda through life table analysis, while providing enhancements for utilizing substerilizing gamma dose in a modified F1 sterility technique. © 2023 Society of Chemical Industry.

Differential susceptibilities of Brontispa longissima (Coleoptera: Hispidae) to insecticides in Southeast Asia.

To present the susceptibility of Brontispa longissima (Gestro) (Coleoptera: Hispidae) to insecticides, 22 populations were collected in Southeast Asia from 2007 to 2010. Moreover, the laboratory susceptible strain was maintained. The results demonstrated that the lethal concentration50 value of the susceptible strain to avermectin, beta-cypermethrin, and acetamiprid was 0.034, 0.024, and 1.843 mg liter(-1), respectively. Compared with the susceptible strain, 10 populations developed a medium level of resistance to avermectin (10.8-fold < resistance ratio [RR] < 34.7-fold) and 11 populations had low or minor resistance (3.6-fold < RR < 9.6-fold), only one population from Qionghai still remained susceptible (RR = 2.04-fold). The Zhangjiang population expressed high resistance (RR = 46.1-fold), five populations expressed medium resistance (10.6-fold < RR < 18.1-fold), and 12 populations had low resistance (5.36-fold < RR < 9.66-fold) for beta-cypermethrin, only four populations maintained low resistance or susceptibility (2.08-fold < RR< 3.49-fold). All populations were susceptible to acetamiprid (1.65-fold < RR < 2.89-fold), except for the Jakarta population that developed minor resistance (4.33-fold). This study shows that the resistance to beta-cypermethrin and avermectin in B. longissima is widespread. However, most populations remain susceptible to acetamiprid. Acetamiprid may be considered an available option for the control of B. longissima and should be considered when designing pest management programs.

A taxonomic review of the genus Axinoscymnus Kamiya, with descriptions of three new species (Coleoptera, Coccinellidae).

Eleven species of the genus Axinoscymnus Kamiya are revised, and additional three new species are described: Axinoscymnus pingxiangicus Peng et Chen sp. n., Axinoscymnus gongxinensis Peng et Chen sp. n. and Axinoscymnus hamulatus Peng et Chen sp. n. The genus Axinoscymnus is recorded from Nepal for the first time. Several species are also newly reported from the following countries: Axinoscymnus puttarudriahi Kapur et Munshi for China, Laos and Nepal, Axinoscymnus macrosiphonatus Hong for China and Laos, Axinoscymnua navicularis Ren et Pang for Laos and Nepal, Axinoscymnus cardilobus Ren et Pang and Axinoscymnus nigripennis Kamiya for Laos. Nomenclatural history, diagnoses, detailed description, illustrations, and distribution for most species have been provided. A key to the species of this genus is also given.

Potential of Entomopathogenic Nematode HbSD as a Candidate Biocontrol Agent against Spodoptera frugiperda.

Spodoptera frugiperda is a highly destructive and polyphagous pest that causes severe damage to various crops, especially maize. The wide use of chemical insecticides to control S. frugiperda results in resistance against commonly used chemicals and resistant mutations will expand in populations accompanied by a spread to vulnerable areas. Consequently, more effective and friendly strategies must be explored to minimize losses caused by S. frugiperda. Entomopathogenic nematodes (EPN) are good candidates for the biological control of different species of insect pests, including S. frugiperda. In the current study, the infective capabilities of the EPN species HbSD, belonging to Hetrerorhabditis bacteriophora, were evaluated against S. frugiperda under laboratory, greenhouse and field conditions. In laboratory assays, HbSD was highly virulent against 3rd/5th instar larvae, which was related to HbSD concentration and exposure durations. In greenhouse assays, spraying aqueous HbSD also showed good performance in killing larvae on maize leaves. However, the virulence of HbSD decreased in field trials where many adverse factors affecting survival and efficacy were encountered by HbSD. Overall, our study provides an alternative EPN for the biological control of S. frugiperda with the potential to be developed as a sustainable option for efficient pest management.

A review of the genus Sasajiscymnus Vandenberg, 2004 from China (Coleoptera, Coccinellidae).

In the present study, the taxonomy of the genus Sasajiscymnus Vandenberg, 2004 from China is revised. The number of Chinese species of Sasajiscymnus was increased from thirty-five to sixty-four. Twenty-seven species are newly described: S. fusinus sp. nov., S. acutus sp. nov., S. cultratus sp. nov., S. subulatus sp. nov., S. lishanicus sp. nov., S. striatus sp. nov., S. wuliangshan sp. nov., S. aureus sp. nov., S. longus sp. nov., S. latus sp. nov., S. obliquus sp. nov., S. niganulus sp. nov., S. maculatus sp. nov., S. variabilis sp. nov., S. flexus sp. nov., S. sagittalis sp. nov., S. robustus sp. nov., S. atypicus sp. nov., S. intricatus sp. nov., S. pseudoamplus sp. nov., S. flavostictus sp. nov., S. falcatus sp. nov., S. parallelus sp. nov., S. tumidus sp. nov., S. guniujiang sp. nov., S. applanatus sp. nov., S. cuonaicus sp. nov. Two species are newly recorded from China: S. ocelliferus (Canepari, 1997) and S. nepalicus (Miyatake, 1985). Pseudoscymnus hamatus (Yu & Pang, 1993) is transferred to Sasajiscymnus (comb. nov.). Five species groups of Chinese Sasajiscymnus are proposed for the first time: S. sylvaticus species group, S. hareja species group, S. atypicus species group, S. amplus species group and S. kurohime species group. Keys to the species groups of Sasajiscymnus, as well as to species level within each species group are provided. Furthermore, maps of the species distribution and detailed descriptions to fifty-nine species are also given.

Characterization of the complete mitochondrial genome and phylogenetic analysis of Menochilus sexmaculata (Fabricius, 1781) (Coleoptera: Coccinellidae).

We sequenced the complete mitochondrial genome of Menochilus sexmaculata (Fabricius, 1781) and compared it with that of other insects. The mitogenome of M. sexmaculata is a circular molecule of 16,663 bp with 75.00% AT content, containing 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and one non-coding control region. All of the PCGs use the typical ATN as the initiation codon, with the exception of cox1 and nad3 which begin with AAT and TTG, respectively. Cox1, cox2, cox3, nad3, nad4, nad5 and nad6 employ a single T as a termination signal, while others have the typical termination codons (TAA or TAG). All the 22 typical animal tRNA genes are found in M. sexmaculata mitogenome, and most of the tRNAs could be folded into the classic cloverleaf secondary structure. Phylogenetic tree based on 13 PCGs suggested that M. sexmaculata is closely related to Anatis ocellata and Calvia championorum, and clustered within Coccinellidae.

Full-length transcriptome analysis of Spodoptera frugiperda larval brain reveals detoxification genes.

BACKGROUND: Spodoptera frugiperda (J. E. Smith), commonly known as fall armyworm (FAW), is one of the most destructive agricultural pests in the world and has posed a great threat to crops. The improper use of insecticides has led to rapid development of resistance. However, the genetic data available for uncovering the insecticide resistance mechanisms are scarce. METHODS: In this study, we used PacBio single-molecule real-time (SMRT) sequencing aimed at revealing the full-length transcriptome profiling of the FAW larval brain to obtain detoxification genes. RESULTS: A total of 18,642 high-quality transcripts were obtained with an average length of 2,371 bp, and 11,230 of which were successfully annotated in six public databases. Among these, 5,692 alternative splicing events were identified.

Comparative Performance of the Fall Armyworm (Lepidoptera: Noctuidae) Reared on Various Cereal-Based Artificial Diets.

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), is a polyphagous and highly destructive agricultural pest that invaded mainland China in 2019. To facilitate research on this pest, it is important to formulate and formalize a suitable artificial diet based on local ingredients. In this study, the life histories of fall armyworm reared on corn leaves and four artificial diets were recorded. The four artificial diets used were: soybean and sucrose-based (SS), soybean and wheat germ-based (SW), chickpea and wheat germ-based (CPW), and corn and soybean-based (CNS). The intrinsic rates of increase were 0.1957, 0.1981, 0.1816, 0.1748, and 0.1464 per day in the fall armyworm populations fed corn leaves, CNS, SW, CPW, and SS, respectively. The highest fecundity (F = 1225.4 eggs per female) and net reproduction rate (R0 = 544.7 offspring per individual) were observed for the fall armyworm reared on the CNS diet. Moreover, the developmental rate, survival rate, and fecundity were used to calculate the projection of the population growth. Projection results showed that the fall armyworm populations can increase considerably faster when fed the CNS diet compared with the other diets. In addition, the mass-rearing system showed that the most efficient and economical strategy would be to rear the fall armyworm on the CNS diet. The results indicated that the CNS diet was the most suitable diet for the fall armyworm mass rearing.

Development of Single Nucleotide Polymorphism (SNP) Markers for Analysis of Population Structure and Invasion Pathway in the Coconut Leaf Beetle Brontispa longissima (Gestro) Using Restriction Site-Associated DNA (RAD) Genotyping in Southern China.

To determine population genomic structure through high-throughput sequencing techniques has revolutionized research on non-model organisms. The coconut leaf beetle, Brontispa longissima (Gestro), is a widely distributed pest in Southern China. Here, we used restriction site-associated DNA (RAD) genotyping to assess the invasion pathway by detecting and estimating the degree of genetic differentiation among 51 B. longissima accessions collected from Southern China. A total of 10,127 SNPs were obtained, the screened single nucleotide polymorphism (SNP) information was used to construct the phylogenetic tree, FST analysis, principal component analysis, and population structure analysis. Genetic structure analysis was used to infer the population structure; the result showed that all accessions were divided into Hainan population and non-Hainan population. The Hainan population remained stable, only the Sansha population differentiated, and the non-Hainan populations have gradually differentiated into smaller sub-populations. We concluded that there are two sources of invasion of B. longissima into mainland China: Taiwan and Hainan. With the increase of the invasion time, the Hainan population was relatively stable, and the Taiwan population was differentiated into three sub-populations. Based on the unrooted phylogenetic tree, we infer that Taiwan and Hainan are the two invasive base points. The Taiwan population invaded Fujian, Guangdong, and Guangxi, while the Hainan population invaded Yunnan and Sansha. Our results provide strong evidence for the utility of RAD sequencing (RAD-seq) in population genetics studies, and our generated SNP resource could provide a valuable tool for population genomics studies of B. longissima in the future.

Inter-country trade, genetic diversity and bio-ecological parameters upgrade pest risk maps for the coconut hispid Brontispa longissima.

BACKGROUND: Invasions of a number of tree-feeding beetles have increased globally and pose a mounting threat to the world's trees, production forests and natural habitats. An in-depth understanding of the determinants of invasion potential of a given species and invasibility of novel environments can help forecast future invasions and avert undesirable socio-economic impacts. Here, we quantitatively assess the (multivariate) drivers of historic invasions of the coconut hispid Brontispa longissima (Coleoptera: Chrysomelidae) across the Asia-Pacific region and critically assess its invasion potential for other key coconut-growing regions. RESULTS: Genetic variation of B. longissima in its invaded range indicated multiple incursions, likely associated with (short-range) natural dispersal and (long-range) trade in ornamental palms and coconut plantlets. Interception records at China's ports of entry accentuate the role of traded planting material. The high fecundity and prolonged, yet adaptable, oviposition period of B. longissima further enhance the invasiveness of this species and aid its successful establishment. Coconut-growing areas are identified with high climatic suitability for B. longissima, and where strengthened biosecurity protocols can prevent future invasions. CONCLUSION: A combined assessment of inter-country trade patterns, population genetics and species bio-ecology (e.g. climate-related development) illuminates the dispersal pathways of invasive species, assesses invasibility of particular geographies, guides quarantine interventions and thus can effectively avert future invasions. © 2019 Society of Chemical Industry.

Combination of label-free quantitative proteomics and transcriptomics reveals intraspecific venom variation between the two strains of Tetrastichus brontispae, a parasitoid of two invasive beetles.

The venom apparatus is a conserved organ in parasitoids that shows adaptations correlated with life-style diversification. Combining transcriptomics and label-free quantitative proteomics, here we explored the venom apparatus components of the endoparasitoid Tetrastichus brontispae (Eulophidae), and provide a comparison of the venom apparatus proteomes between its two closely related strains, T. brontispae-Octodonta nipae (Tb-On) and T. brontispae-Brontispa longissima (Tb-Bl). Tb-Bl targets the B. longissima pupa as its habitual host. However, Tb-On is an experimental derivative of Tb-Bl, which has been exposed to the O. nipae pupa as host consecutively for over 40 generation. Results showed that approximately 1505 venom proteins were identified in the T. brontispae venom apparatus. The extracts contained novel venom proteins, such as 4-coumarate-CoA ligase 4. A comparative venom proteome analysis revealed that significant quantitative and qualitative differences in venom composition exist between the two strains; although the most abundant venom proteins were shared between them. The differentially produced proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response. Six of these enriched proteins presented increased levels in Tb-On, and this result was validated by parallel reaction monitoring (PRM) analysis. Overall, our data reveal that venom composition can evolve quickly and respond to host selection.

Antennal and abdominal transcriptomes reveal chemosensory gene families in the coconut hispine beetle, Brontispa longissima.

Antennal and abdominal transcriptomes of males and females of the coconut hispine beetle Brontispa longissima were sequenced to identify and compare the expression patterns of genes involved in odorant reception and detection. Representative proteins from the chemosensory gene families likely essential for insect olfaction were identified. These include 48 odorant receptors (ORs), 19 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 34 odorant binding proteins (OBPs) and 16 chemosensory proteins (CSPs). Phylogenetic analysis revealed the evolutionary relationship of these proteins with homologs from Coleopterans or other insects, and led to the identification of putative aggregation pheromone receptors in B. longissima. Comparative expression analysis performed by calculating FPKM values were also validated using quantitative real time-PCR (qPCR). The results revealed that all ORs and antennal IRs, two IR co-receptors (BlonIR8a and BlonIR25a) and one SNMP (BlonSNMP1a) were predominantly expressed in antennae when compared to abdomens, and approximately half of the OBPs (19) and CSPs (7) were enriched in antennae. These findings for the first time reveal the identification of key molecular components in B. longissima olfaction and provide a valuable resource for future functional analyses of olfaction, and identification of potential targets to control this quarantine pest.

Antifeedant activity of spin-labeled derivatives of deoxypodophyllotoxin against Brontispa longissima larvae.

To continue our search for natural product-based compounds for the control of Brontispa longissima larvae, eight spin-labeled podophyllotoxin derivatives (4a-h) and the intermediates 2 and 3 were tested for their insect antifeedant activity against third-instar larvae of B. longissima. Among all the tested compounds, 4a, 4c, 2 and 3 showed pronounced antifeedant activities with AFC50 values of 0.16, 0.31, 0.15 and 0.28 mg/mL, respectively. The different antifeedant activity ranges of these compounds indicated that variation of the structures of L-amino acids in these compounds markedly affected the activity profiles of this compound class, and some important SAR information has been revealed from it.