Mori Cells Research Articles

Production of porcine parvovirus virus-like particles using silkworm larvae

Abstract Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.

Identification of LARK as a novel and conserved G-quadruplex binding protein in invertebrates and vertebrates.

Double-stranded DNAs are usually present in the form of linear B-form double-helix with the base pairs of adenine (A) and thymine (T) or cytosine (C) and guanine (G), but G-rich DNA can form four-stranded G-quadruplex (G4) structures, which plays important roles in transcription, replication, translation and protection of telomeres. In this study, a RNA recognition motif (RRM)-containing protein, BmLARK, was identified and demonstrated to bind G4 structures in the promoters of a transcription factor BmPOUM2 and other three unidentified genes of Bombyx mori, as well as three well-defined G4 structures in the human genes. Homologous LARKs from Bombyx mori, Drosophila melanogaster, Mus musculus and Homo sapiens bound G4 structures in BmPOUM2 and other genes in B. mori and H. sapiens. Upon binding, LARK facilitated the formation and stability of the G4 structure, enhancing the transcription of target genes. The G4 structure was visualized in vivo in cells and testis from invertebrate B. mori and vertebrate Chinese hamster ovary (CHO) cells. The results of this study strongly suggest that LARK is a novel and conserved G4-binding protein and that the G4 structure may have developed into an elaborate epigenetic mechanism of gene transcription regulation during evolution.

BmGeminin2 interacts with BmRRS1 and regulates Bombyx mori cell proliferation

ABSTRACTGeminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents re-replication in vertebrates and invertebrates. Previously, we identified two Geminin genes, BmGeminin1 and BmGeminn2, in the silkworm Bombyx mori, and we found that RNA interference of BmGeminin1 led to re-replication. However, the function of BmGeminin2 remains poorly understood. In this study, we found that knockdown of BmGeminin2 can improve cell proliferation, and upregulated G2/M-associated gene-cyclinB/CDK1 expression. Then, we performed yeast two-hybrid screening to identify interacting proteins. Our results yielded 23 interacting proteins, which are involved in DNA replication, chromosome stabilization, embryonic development, energy, defense, protein processing, or structural protein. Here, we focused on BmRRS1, a chromosome congression-related protein that is closely related to cell cycle G2/M progression. The interaction between BmGeminin2 and BmRRS1 was confirmed by immunofluorescence and immunoprecipitation. Analysis of its expression profile showed that BmRRS1 was related to BmGeminin2. In addition, BmGeminin2 overexpression downregulated the BmRRS1 transcript. Knockdown of BmGeminin2 led to upregulation of the BmRRS1 transcript. Furthermore, overexpression of BmRRS1 can upregulate G2/M-associated gene-cyclinB/CDK1 expression, and improved cell proliferation, consistent with the effects of BmGeminin2 knockout. In addition, BmRRS1 RNA interference can eliminate the impact of BmGem2 knockout on cell proliferation, the ratio of cell cycle stage and the expression of cyclinB/CDK1. These data suggested that the cell proliferation advantage of BmGeminin2 knockout was closely related to BmRRS1. Our findings provide insight into the functions of Geminin and the mechanisms underlying the regulation of the cell cycle in the silkworm.

Transcriptional repression of endogenous genes in BmE cells using CRISPRi system

Recent advancements in genetic engineering technology have led to the development of CRISPR interference (CRISPRi) as a precise tool for regulating gene expression. When CRISPR/dCas9 is fused with transcriptional repressors, the system can robustly silence endogenous gene expression. The CRISPR/Cas9 tool is a promising alternative in organisms (e.g., Bombyx mori) that do not respond to traditional gene suppression techniques, such as RNA interference (RNAi). However, transcriptional repressors remain poorly categorized in multiple cell types and species, leading to difficulties in optimizing performance and efficiency. Here, we tested CRISPRi usability and efficiency in Bombyx mori cells (BmE). We fused dCas9 to five transcriptional repressors including KRAB, Hairy, SID, SRDX, and ERD. All five constructs were efficient in BmE cells. In a proof-of-concept experiment, we showed that CRISPRi acting on BmSoxE (a gene involved in cell proliferation) could generate similar phenotypes as RNAi gene suppression. Moreover, CRISPRi has fewer off-target effects. Through co-transfection of BmE cells with sgRNAs, we also demonstrated that dCas9 could simultaneously repress the expression of multiple genes. Furthermore, we identified sgRNA distance from transcriptional start site (TSS) and the dCas9: sgRNA ratio as the two limiting factors of CRISPRi efficiency. Our results demonstrated that CRISPR/dCas9 is a viable and rapid alternative for functional investigations of the B. mori genome and perhaps other Lepidoptera insects.

Inhibition of Period Gene Expression Causes Repression of Cell Cycle Progression and Cell Growth in the Bombyx mori Cells.

Circadian clock system disorders can lead to uncontrolled cell proliferation, but the molecular mechanism remains unknown. We used a Bombyx mori animal model of single Period gene (BmPer) expression to investigate this mechanism. A slow growing developmental cell model (Per-KD) was isolated from a B. mori ovarian cell line (BmN) by continuous knock down of BmPer expression. The effects of BmPer expression knockdown (Per-KD) on cell proliferation and apoptosis were opposite to those of m/hPer1 and m/hPer2 in mammals. The knockdown of BmPer expression led to cell cycle deceleration with shrinking of the BmN cell nucleus, and significant inhibition of nuclear DNA synthesis and cell proliferation. It also promoted autophagy via the lysosomal pathway, and accelerated apoptosis via the caspase pathway.

Phosphorylation of nucleopolyhedrovirus 39K is essential for the regulation of viral gene transcription in silkworm cells.

39K protein was proven to be the most notable hyperphosphorylated protein of Bombyx mori nucleopolyhedrovirus (BmNPV), since its amino acid 136 has the highest phosphorylation ratio (16.683) among all of BmNPV phosphorylated proteins (Shobahah et al., 2017). Therefore, we inserted positive (mimicking phosphorylation) and negative (mimicking dephosphorylation) mutants of the highest phosphorylated site of 39K and the wild type 39k into the 39k-ko-Bacmid using the Bac-to-Bac system. These three kinds of recombinant Bacmids along with wild type and knocked-out Bacmid were then transfected into the Bombyx mori cells (BmN) and further investigated by qPCR analysis. The results of the qPCR showed that 39K phosphorylation had no significant effect on viral genome replication, unlike the positive mutation that reduced the viral gene transcription drastically compared to the wild type. Thus, phosphorylation of BmNPV 39K protein does not appear to be the essential mechanism for viral genome replication, even though it has an important role in the viral gene transcription. Keywords: BmNPV; 39K; phosphorylation; virus replication; virus transcription.

Antiviral immune responses of Bombyx mori cells during abortive infection with Autographa californica multiple nucleopolyhedrovirus

Lepidopteran cells rely on multiple antiviral responses to defend against baculovirus infections, including apoptosis, global protein synthesis shutdown, and rRNA degradation. Here, we characterized apoptosis and rRNA degradation in Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Bombyx mori cells, a system resulting in abortive infection, in relation to viral DNA replication and viral late gene expression. RNAi-mediated silencing of viral DNA replication-related genes prevented apoptosis, but not rRNA degradation, in B. mori cells infected with p35-deficient AcMNPV. Additionally, AcMNPV, but not B. mori nucleopolyhedrovirus (BmNPV), drastically reduced B. mori cellular iap1 transcript levels and p35-deficient AcMNPV induced more prominent apoptosis than did p35-deficient BmNPV. These results, together with previous results that global protein synthesis shutdown follows viral DNA replication, demonstrate that rRNA degradation is the primary antiviral response that abolishes productive AcMNPV infection of B. mori cells. Our results also demonstrate that B. mori cells induce apoptosis to a different extent depending on NPV species.

Stimulator of interferon genes (STING) provides insect antiviral immunity by promoting Dredd caspase–mediated NF-κB activation

The antiviral cGMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. In this study, we found that cGAMP is produced in silkworm (Bombyx mori) cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, we identified BmSTING, a potential cGAMP sensor in B. mori We observed that BmSTING overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated BmSTING knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-κB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of B. mori larvae. Moreover, we identified a caspase-8-like protein (BmCasp8L) as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. Our findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect B. mori and have identified several BmSTING-interacting proteins that control antiviral defenses.

Construction of a Bombyx mori cell line that stably express the susceptible gene +nsd-2 of Bombyx mori bidensovirus.

Bombyx mori bidensovirus (BmBDV) is the only type species established in the new genusBidensovirus within the new family Bidnaviridae. The BmBDV infected silkworm midgut shows symptoms such as the nucleus of midgut columnar cells expanded. It is confirmed theBmBDV-sensitive gene +nsd-2, which encoding an amino acid transporter protein might be the receptor of BmBDV. The deletion of this gene causes the silkworm resistance to BmBDV infection. There is no permissive cell line for BmBDV infection has been established yet. The cell lines (BmN, etc.) are used broadly for silkworm viruses study are mainly resistant to BmBDV infection. Therefore, the establishment of BmBDV permissive cell line is particularly important for studying BmBDV in vitro. In this study, we successfully constructed a cell line BmN (+nsd-2), which expressed +nsd-2 gene stably. The NSD-2 expression and localization could be detected in BmN (+nsd-2 ) cells via RT-PCR, western blot and immunofluorescence. The interaction between NSD-2 and the major structural protein VP of BmBDV was determined by Co-IP. However, we failed to detect the replication of BmBDV. This cell lines constructed has the potential for identifying the protein binding to the NSD-2 and provide a basis for study of BmBDV co-receptors and invasion mechanism.

Bombyx mori homolog of tumor suppressor p53 is involved in apoptosis-mediated antiviral immunity of B. mori cells infected with nucleopolyhedrovirus

Apoptosis is important in antiviral immunity and affects viral multiplication and pathogenesis. Here, we showed that Bombyx mori cells transiently expressing B. mori homolog of the tumor suppressor p53 (Bm-p53) protein underwent apoptosis accompanied by elevated caspase-3-like protease activity and processing of B. mori Dronc (Bm-Dronc). RNAi-mediated silencing of bm-p53 expression, which significantly diminished accumulation of bm-p53 transcript and Bm-p53 protein, prevented apoptosis of B. mori cells infected with a recombinant B. mori nucleopolyhedrovirus defective in the anti-apoptotic p35 gene (vBmΔp35) and abolished the activation of caspase-3-like protease and processing of Bm-Dronc. Apoptosis in vBmΔp35-infected B. mori cells is associated with viral DNA replication, suggesting involvement of the DNA damage response. The Bm-p53 pro-apoptotic function is also found in Spodoptera frugiperda and Lymantria dispar cells. These results indicate that apoptosis induction in vBmΔp35-infected B. mori cells is a Bm-p53-mediated process promoted by the commencement of viral DNA replication.

Identification of a novel host protein SINAL10 interacting with GP64 and its role in Bombyx mori nucleopolyhedrovirus infection

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important pathogen of Bombyx mori, silkworm and causes severe losses in the silk industry. During the virus infectious cycle, budded virus (BVs) and occlusion-derived virus (ODVs) particles, which have identical genetic content but different phenotypes, are produced. The envelope glycoprotein GP64, specific in BVs, is involved in host cell receptor binding and is sufficient to mediate membrane fusion during the viral entry. However, the host cell factors, interacting with GP64 to mediate BVs infection, are still unknown. In this study, a cDNA library of Bombyx mori cells (BmN) was constructed and yeast two-hybrid screening was used to identify the host cell factors interacting with GP64. One of the eight candidate proteins encoded the E3 ubiquitin-protein ligase SINA-like 10 (SINAL10), was further confirmed through coimmunoprecipitation assays as novel GP64 binding protein. Moreover, overexpression of SINAL10 significantly enhances viral reproduction, and conversely, silencing its expression by small interfering RNAs showed significant inhibitory effects. Collectively, we demonstrated that SINAL10 is a novel GP64-binding protein that stimulates BmNPV proliferation.

Analyses of the transcriptome of Bombyx mori cells infected with either BmNPV or AcMNPV

Baculoviruses are insect-specific DNA viruses with a restricted host range. Two examples of such include Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). AcMNPV is a commercially available and widely used baculovirus prototype which can infect 39 species in 13 families. On the other hand, BmNPV is a major pathogen of silkworms which has developed high host specificity to Bombyx mori. Coincidentally, AcMNPV and BmNPV are highly homologous, but share no overlapping host range on a genomic level. In fact, these two similar viruses have extremely different infection outcomes in Bombyx mori. We hypothesized that the determination of host specificity may depend on virus-host interactions and several genes may be involved in determining host specificity. Therefore, we used next generation sequencing (NGS) to analyze transcriptome responses of hosts to these viruses. A transcriptome library was constructed, annotated, and grouped after sequence assembly. The comparison of gene expressions showed several significant differences in the gene expression profiles of BmNPV and AcMNPV, especially in cases where genes involved with immune responses were verified by RNA interference. In addition, studies have shown that small RNA plays a role in virus-host interaction, and further determines host specificity. Therefore, we screened microRNA induced by AcMNPV infection, and then combined NGS data from cellular gens to predict possible regulation networks. The manipulation of virus-host specificity can provide a breakthrough for the application of baculovirus in protein expression systems and the development of bio-control agents.

Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus

BackgroundBombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive.MethodEmploying tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined.ResultsTotally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D.ConclusionThe phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

Inhibition of expression of the circadian clock gene Period causes metabolic abnormalities including repression of glycometabolism in Bombyx mori cells

Abnormalities in the circadian clock system are known to affect the body’s metabolic functions, though the molecular mechanisms responsible remain uncertain. In this study, we achieved continuous knockdown of B. mori Period (BmPer) gene expression in the B. mori ovary cell line (BmN), and generated a Per-KD B. mori model with developmental disorders including small individual cells and slow growth. We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism. Amino acids that used glucose metabolites as a source were also down-regulated, while lipid metabolism and nucleotide metabolism were significantly up-regulated. Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism, as well as to metabolites such as aspartate, alanine, and xanthine in other pathways. Further validation experiments showed that the activities of the key enzymes of glucose metabolism, hexokinase, phosphofructokinase, and citrate synthase, were significantly decreased and transcription of their encoding genes, as well as that of pyruvate kinase, were also significantly down-regulated. We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism, and may be associated with changes in cellular amino acid metabolism, and in cell growth and development.

Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.

RRNA degradation in Bombyx mori and Bombyx mandarina cells infected with heterologous nucleopolyhedroviruses

rRNA degradation in Bombyx mori and Bombyx mandarina cells infected with heterologous nucleopolyhedroviruses

A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution.

Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV), have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143), but not BmNPV-P143 (BmP143) or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV.

Important roles played by TGF-β member of Bmdpp and Bmdaw in BmNPV infection.

Transforming growth factor (TGF)-β superfamily members inhibit Bombyx mori nucleohedrovirus (BmNPV) multiplication in silkworm are not determined. In this study, we first found that BmNPV RNA transcription and protein expression level were regulated by TGF-β members, Decapentaplegic (Bmdpp) and Dawdle (Bmdaw) in the domesticated silkworm, B. mori and silkworm ovary-derived cells. Furthermore, subcellular localization showed that Bmdpp and Bmdaw were mainly presented in cytomembrane of the cultured BmN cells. Tissues expression pattern analysis found that the highest expression levels of Bmdpp and Bmdaw genes were in the hemocyte of fifth instar larvae. During the immune response, the expression level of Bmdpp gene was elevated and Bmdaw gene was declined in BmNPV infected BmN cells and silkworm. The multiplication of BmNPV was inhibited by overexpression of Bmdpp and Bmdaw genes in BmN cells. RNA interference experiments found that the multiplication of BmNPV was raised with specific siRNAs of Bmdpp and Bmdaw genes in BmN cells. The antiviral immune pathways were not significantly regulated by the TGF-β superfamily members. Taken together, these findings provided a clue to understand the function of Bmdpp and Bmdaw gene in response to the BmNPV infection in silkworm.

Suppression of Bm-Caspase-1 Expression in BmN Cells Enhances Recombinant Protein Production in a Baculovirus Expression Vector System

Baculovirus expression vector systems (BEVSs) have been widely used for recombinant protein production. Many studies have tried to improve the recombinant protein production by either modification of BEVSs vectors or by cell line selection. In this study, using a modified shRNA vector, we established a stable Bombyx mori cell line that significantly inhibits expression of caspase-1 after selection. We further compared cell proliferation and viability between caspase-1-suppressed cells and control cells, and found that there is no significant difference in these stable cell lines. We utilized these cell lines to analyze recombinant protein production after infection with recombinant baculovirus. We found that both intracellular and extracellular recombinant protein production significantly increased when expression of caspase-1 is inhibited in BmN cells. These data indicate that blocking the apoptotic pathway is a promising way to enhance recombinant protein production in BEVSs.

Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.