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1.
Proc Natl Acad Sci U S A ; 119(15): e2201071119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377802

RESUMO

Significance The genome of insects with complete metamorphosis contains the instructions for making three distinct body forms, that of the larva, of the pupa, and of the adult. However, the molecular mechanisms by which each gene set is called forth and stably expressed are poorly understood. A half century ago, it was proposed that there was a set of three master genes that inhibited each other's expression and enabled the expression of genes for each respective stage. We show that the transcription factor chinmo is essential for maintaining the larval stage in Drosophila, and with two other regulatory genes, broad and E93, makes up the trinity of mutually repressive master genes that underlie insect metamorphosis.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteínas do Tecido Nervoso , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/metabolismo , Metamorfose Biológica/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Pupa/genética , Pupa/metabolismo
2.
Int J Mol Sci ; 23(5)2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35269892

RESUMO

Allatostatin C (PISCF/AST) is a neuropeptide gene that affects juvenile hormone (JH) synthesis in the corpora allata. Juvenile hormone acid O-methyltransferase (JHAMT) is a key gene in the JH biosynthetic pathway. In this study, two genes encoding DaAST and DaJHAMT were cloned. Both DaAST and DaJHAMT were expressed in the larvae, pupae and adults of Chinese white pine beetle (Dendroctonus armandi), and highly expressed in the head and the gut. The expression of the two genes was induced by JH analog (JHA) methoprene and the functions of the two genes were then investigated by RNAi. Considering the role of hormones in metamorphosis, JHA significantly induced DaAST and DaJHAMT in the larval stage. DaAST knockdown in larvae, pupae and adults significantly increased the DaJHAMT mRNA levels. Moreover, knockdown of DaAST instead of DaJHAMT increased pupae mortality and the abnormal rate of emergence morphology and reduced emergence rates. However, knockdown of DaJHAMT instead of DaAST significantly reduced frontalin biosynthesis in adult males. The results showed that DaAST acts as an allatostatin and inhibits JH biosynthesis, and that JHAMT is a key regulatory enzyme for JH synthesis in the D. armandi.


Assuntos
Hormônios Juvenis , Neuropeptídeos , Animais , Corpora Allata/metabolismo , Hormônios Juvenis/metabolismo , Larva/metabolismo , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Pupa/genética , Pupa/metabolismo
3.
Int J Biol Macromol ; 206: 422-434, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35245573

RESUMO

C-type lectins (CTLs) play vital roles in invertebrates' innate immunity. Six CTL-X type lectins are identified in Tribolium castaneum. However, their functions and regulating mechanisms remain elusive. Here, TcCTL12, one CTL-X, was identified and cloned from T. castaneum. Spatiotemporal expression profiling revealed that TcCTL12 highly expressed in late pupa and early adult of T. castaneum in comparison with other developmental stages, and exhibited the highest expression level in the haemolymph and central nervous system (CNS). Then, the expression of TcCTL12 was remarkably induced by the stimulation of Escherichia coli and Staphylococcus aureus. Moreover, the recombinant protein TcCTL12 could bind pathogen-associated molecular patterns (PAMPs) including LPS and PGN, and displayed agglutinative activity to both Gram-positive and Gram-negative bacteria in a calcium-dependent manner in vitro. Furthermore, RNAi of TcCTL12 caused T. castaneum pupation and eclosion defected. The abnormal pupa thinned their epidermal, and appeared the abnormal development of muscle cell compared with the control group. Additionally, depletion of TcCTL12 resulted in reducing fertility of offspring and affected their fecundity. In sum, these results indicated that TcCTL12 had extensive functions in the regulation of development in T. castaneum, in addition to the immune response. It further expanded insights into CTL functions in insects.


Assuntos
Tribolium , Animais , Antibacterianos/metabolismo , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Imunidade Inata , Pupa/metabolismo , Tribolium/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(11): e2118871119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35259020

RESUMO

SignificanceJuvenile hormone (JH), a sesquiterpenoid, regulates many aspects of insect development, including maintenance of the larval stage by preventing metamorphosis. In contrast, ecdysteroids promote metamorphosis by inducing the E93 transcription factor, which triggers apoptosis of larval cells and remodeling of the larval midgut. We discovered that JH suppresses precocious larval midgut-remodeling by inducing an epigenetic modifier, histone deacetylase 3 (HDAC3). JH-induced HDAC3 deacetylates the histone H4 localized at the promoters of proapoptotic genes, resulting in the suppression of these genes. This eventually prevents programmed cell death of midgut cells and midgut-remodeling during larval stages. These studies identified a previously unknown mechanism of JH action in blocking premature remodeling of the midgut during larval feeding stages.


Assuntos
Aedes/fisiologia , Apoptose , Sistema Digestório/metabolismo , Histona Desacetilases/metabolismo , Hormônios Juvenis/metabolismo , Animais , Apoptose/genética , Sistema Digestório/anatomia & histologia , Ecdisona/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desacetilases/genética , Histonas/metabolismo , Larva , Pupa/metabolismo
5.
J Agric Food Chem ; 70(12): 3862-3871, 2022 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-35230117

RESUMO

This study aimed at exploring dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from silkworm pupae proteins by in silico analysis and in vitro assessments. In silico analysis of 274 silkworm pupae proteomes indicated that DPP-IV inhibitory peptides can be released from silkworm pupae proteins. In vitro assessments revealed that pepsin and bromelain led to better production of DPP-IV inhibitory peptides from silkworm pupae protein. Notably, peptide fractions (<1 kDa) from pepsin- and bromelain-treated hydrolysates exhibited more potent DPP-IV inhibitory activities. Two novel DPP-IV inhibitory peptides (Leu-Pro-Pro-Glu-His-Asp-Trp-Arg and Leu-Pro-Ala-Val-Thr-Ile-Arg) were identified by LC-MS/MS with IC50 values of 261.17 and 192.47 µM, respectively. Enzyme kinetics data demonstrated that these two peptides displayed a mixed-type DPP-IV inhibition mode, which was further validated by molecular docking data. Overall, in silico analysis combined with in vitro assessments can serve as an effective and rapid approach for discovery of DPP-IV peptides from silkworm pupae proteins.


Assuntos
Bombyx , Inibidores da Dipeptidil Peptidase IV , Animais , Bombyx/metabolismo , Cromatografia Líquida , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Simulação de Acoplamento Molecular , Peptídeos/química , Pupa/metabolismo , Espectrometria de Massas em Tandem
6.
J Mol Biol ; 434(5): 167456, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35045329

RESUMO

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.


Assuntos
Borboletas , Quitinases , Animais , Borboletas/metabolismo , Quitina/química , Quitina/metabolismo , Quitinases/metabolismo , Insetos/metabolismo , Pupa/metabolismo
7.
Dev Biol ; 483: 107-111, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35007518

RESUMO

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.


Assuntos
Células Epiteliais/metabolismo , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva/metabolismo , Manduca/metabolismo , Muda/imunologia , Pupa/metabolismo , Animais , Quitina/biossíntese , Epitélio/metabolismo , Hemócitos/metabolismo , Hemolinfa/imunologia , Imunidade Humoral , Larva/imunologia , Manduca/imunologia , Pupa/imunologia , Via Secretória/imunologia , Trealase/metabolismo
8.
Arch Insect Biochem Physiol ; 109(4): e21855, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34811799

RESUMO

Disruption of the presenilin (ps) genes are the major genetic cause of familial Alzheimer's disease. The silkworm, Bombyx mori (B. mori), is an important model insect. The ps homologue gene in B. mori was identified and characterized. However, the role of ps in B. mori was poorly understood. Here, we found that Bmps was ubiquitously expressed in all the tested tissues during metamorphosis. In the current study, loss-of-function analysis of Bmps was performed by the binary transgenic CRISPR/cas9 system. Compared with the wild type, the developmental time of ∆Bmps animals were significantly delayed. In addition, ∆Bmps showed abnormal appendage including antenna, leg, wing and eye during pupal and adult stages. RNA-seq analysis indicated that apoptosis and proliferation related pathways were affected in ∆Bmps. Moreover, the Hippo pathway was affected by Bmps depletion in brain and wing disc. Our results suggest that PS is essential for maintaining the dynamic balance of apoptosis and proliferation during metamorphosis.


Assuntos
Bombyx , Animais , Bombyx/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva , Metamorfose Biológica , Presenilinas/metabolismo , Pupa/genética , Pupa/metabolismo
9.
Cell Rep ; 37(13): 110151, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34965423

RESUMO

Ran's GTPase-activating protein (RanGAP) is tethered to the nuclear envelope (NE) in multicellular organisms. We investigated the consequences of RanGAP localization in human tissue culture cells and Drosophila. In tissue culture cells, disruption of RanGAP1 NE localization surprisingly has neither obvious impacts on viability nor nucleocytoplasmic transport of a model substrate. In Drosophila, we identified a region within nucleoporin dmRanBP2 required for direct tethering of dmRanGAP to the NE. A dmRanBP2 mutant lacking this region shows no apparent growth defects during larval stages but arrests at the early pupal stage. A direct fusion of dmRanGAP to the dmRanBP2 mutant rescues this arrest, indicating that dmRanGAP recruitment to dmRanBP2 per se is necessary for the pupal ecdysis sequence. Our results indicate that while the NE localization of RanGAP is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, we find a requirement for this localization during pupal development.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Pupa/crescimento & desenvolvimento , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HCT116 , Humanos , Chaperonas Moleculares/genética , Membrana Nuclear/genética , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Pupa/genética , Pupa/metabolismo
10.
J Insect Sci ; 21(5)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34718645

RESUMO

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.


Assuntos
Apoptose/efeitos da radiação , Raios gama , Expressão Gênica/efeitos da radiação , Tephritidae/efeitos da radiação , Animais , Antioxidantes/metabolismo , Antioxidantes/efeitos da radiação , Apoptose/genética , Catalase/metabolismo , Catalase/efeitos da radiação , Radioisótopos de Cobalto/farmacologia , Controle de Insetos/métodos , Proteínas de Insetos/metabolismo , Proteínas de Insetos/efeitos da radiação , Larva/genética , Larva/metabolismo , Larva/fisiologia , Larva/efeitos da radiação , Longevidade/efeitos da radiação , Malondialdeído/metabolismo , Malondialdeído/efeitos da radiação , Peroxidase/metabolismo , Peroxidase/efeitos da radiação , Controle de Pragas/métodos , Pupa/genética , Pupa/metabolismo , Pupa/fisiologia , Pupa/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiação , Tephritidae/genética , Tephritidae/metabolismo , Tephritidae/fisiologia
11.
J Invertebr Pathol ; 186: 107674, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34606828

RESUMO

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.


Assuntos
Beauveria/fisiologia , Proteínas de Insetos/genética , Metarhizium/fisiologia , Tribolium/genética , Animais , Proteínas de Insetos/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Pupa/microbiologia , Tribolium/crescimento & desenvolvimento , Tribolium/metabolismo , Tribolium/microbiologia
12.
Development ; 148(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34532737

RESUMO

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Epitélio/metabolismo , Proteínas Repressoras/metabolismo , Asas de Animais/metabolismo , Junções Aderentes/metabolismo , Animais , Proteínas de Transporte , Citoplasma/metabolismo , Morfogênese/fisiologia , Pupa/metabolismo
13.
Proc Natl Acad Sci U S A ; 118(35)2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34429358

RESUMO

Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. Here, we demonstrate that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, our study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bombyx/metabolismo , Proteínas de Drosophila/genética , Drosophila/metabolismo , Ecdisona/biossíntese , Histonas/metabolismo , Proteínas de Insetos/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Larva/metabolismo , Metilação , Pupa/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Esteroides/metabolismo
14.
Pestic Biochem Physiol ; 178: 104934, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34446203

RESUMO

Chitin synthase (CHS) plays a critical role in chitin synthesis and excretion. In most insects, CHSs have been segregated into 1 and 2 classes. CHS1 is responsible for chitin production in the ectodermally-derived epidermal cells. CHS2 is dedicated to chitin biosynthesis in the midgut peritrophic matrix (PM). Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae plants. In this study, we identified HvCHS1 and HvCHS2. We found that HvCHS1 was abundantly transcribed in the larval tracheae and epidermis, whereas HvCHS2 was mainly expressed in the guts. Escherichia coli HT115 expressed double stranded RNAs targeting HvCHS1 and HvCHS2 (dsCHS1 and dsCHS2) were used to immerse potato foliage and the treated leaves were provided to the newly-molted fourth- and third-instar larvae. Ingestion of dsCHS1 by the fourth-instar larvae significantly diminished the target mRNA level and had slight influence on the expression of HvCHS2. In contrast, consumption of dsCHS2 significantly lowered the target mRNA level but triggered the transcription of HvCHS1. Knockdown of HvCHS1, rather than HvCHS2, arrested larval development and impaired larva-pupa-adult transition. A large proportion of HvCHS1 hypomorphs became stunting prepupae, deformed pupae or misshapen adults. Moreover, knockdown of HvCHS1 damaged gut integrity, decreased cuticle thickness, and delayed the formation of newly-generated cuticle layer during ecdysis. Furthermore, depletion of HvCHS1 inhibited the development of trachea system and thinned tracheal taenidia. Ingestion of dsCHS1 at the third-instar stage caused similar but severe negative effects. Our results demonstrated that HvCHS1 is responsible for chitin biosynthesis during ecdysis. Moreover, HvCHS1 is a potential amenable target gene and young larvae are more susceptible to dsRNA.


Assuntos
Quitina Sintase , Besouros , Animais , Quitina/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Besouros/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Muda/genética , Pupa/metabolismo , Interferência de RNA
15.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34417295

RESUMO

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.


Assuntos
Ephemeroptera/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Metamorfose Biológica , Muda , Animais , Ephemeroptera/genética , Ephemeroptera/metabolismo , Proteínas de Insetos/genética , Ninfa/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
16.
J Insect Physiol ; 134: 104294, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34389412

RESUMO

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis.


Assuntos
Drosophila melanogaster , Ecdisona , Triterpenos/farmacologia , Animais , Bombyx/efeitos dos fármacos , Bombyx/metabolismo , Proteínas de Drosophila/efeitos dos fármacos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ecdisona/antagonistas & inibidores , Ecdisona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Juvenis/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Muda/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Extratos Vegetais/farmacologia , Pupa/efeitos dos fármacos , Pupa/crescimento & desenvolvimento , Pupa/metabolismo
17.
Arch Insect Biochem Physiol ; 108(1): e21832, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34250644

RESUMO

Autophagy is a critical mechanism for the self-renewal, proliferation, and differentiation of stem cells. Bombyx mori midgut has stem cells that play a role in the regeneration of the larval epithelium in larval stages and the formation of the pupal midgut epithelium during larval-pupal metamorphosis. In this study, the role of the autophagy mechanism in midgut stem cells during the formation of the pupal midgut was investigated. For this purpose, two different doses of autophagy inhibitor chloroquine were administered to B. mori larvae on days 7 and 8 of the fifth larval stage. Morphological changes during the formation process of the pupal epithelium, expression levels of autophagy-related genes Atg8 and Atg12 in stem cells, and the amounts of lysosomal enzyme acid phosphatase were determined after the application. The obtained findings were evaluated in comparison with the control groups. Abnormalities in the formation of the pupal midgut after inhibition of autophagy showed the significance of the autophagy mechanism during this period.


Assuntos
Autofagia , Bombyx , Intestinos , Metamorfose Biológica/fisiologia , Células-Tronco , Fosfatase Ácida/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Bombyx/citologia , Bombyx/metabolismo , Bombyx/fisiologia , Cloroquina/farmacologia , Intestinos/citologia , Intestinos/efeitos dos fármacos , Larva/citologia , Larva/metabolismo , Pupa/citologia , Pupa/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
18.
Int J Mol Sci ; 22(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34281211

RESUMO

To reveal the mechanism of temperature preference in Tuta absoluta, one of the top 20 plant pests in the world, we cloned and identified TaTRPA1, TaPain, and TaPyx genes by RACE and bioinformatic analysis, and clarified their expression profiles during different development stages using real-time PCR, and revealed their function in preference temperature by RNAi. The full-length cDNA of TaPain was 3136 bp, with a 2865-bp open reading frame encoding a 259.89-kDa protein; and the partial length cDNA of TaPyx was 2326-bp, with a 2025-bp open reading frame encoding a 193.16-kDa protein. In addition, the expression of TaTRPA1 and TaPyx was significantly lower in larvae than other stages, and it was significantly higher in pupae and newly emerging males for TaPain. After feeding target double-stranded RNA (dsRNA), the preferred temperature decreased 2 °C more than the control group. In conclusion, the results firstly indicated the molecular characterization of TRPA subfamily genes and their key role in temperature perception in T. absoluta, and the study will help us to understand the temperature-sensing mechanism in the pest, and will provide some basis for study of other Lepidoptera insects' temperature preference. Moreover, it is of great significance in enriching the research progress of "thermos TRP".


Assuntos
Mariposas/genética , Canal de Cátion TRPA1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Comportamento Animal , Feminino , Larva/metabolismo , Masculino , Mariposas/metabolismo , Pupa/metabolismo , Temperatura
19.
J Med Entomol ; 58(6): 2533-2537, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34302473

RESUMO

Transgenic strains of the mosquito disease vector Aedes aegypti (L.) are being developed for population suppression or modification. Transgenic mosquitoes are identified using fluorescent protein genes. Here we describe DsRed and ZsGreen marker genes driven by the constitutive Ae. aegypti heat shock protein 83 (hsp83) promoter in transgenic mosquitoes. Transgenic larvae and pupae show strong full body expression of the red and green fluorescent proteins. This greatly assists in screening for transgenic individuals while making new or maintaining already established lines. Transient marker gene expression after embryo microinjection was readily visible in developing larvae allowing the separation of individuals that are more likely to produce transgenic offspring. The strongly expressed marker genes developed in this study should facilitate the detection of transgenic Ae. aegypti larvae or pupae in the field.


Assuntos
Aedes/genética , Proteínas de Choque Térmico/genética , Proteínas de Insetos/genética , Proteínas Luminescentes/metabolismo , Regiões Promotoras Genéticas , Aedes/crescimento & desenvolvimento , Aedes/metabolismo , Animais , Animais Geneticamente Modificados , Marcadores Genéticos , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo
20.
Pestic Biochem Physiol ; 176: 104872, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34119217

RESUMO

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.


Assuntos
Locusta migratoria , Tribolium , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Locusta migratoria/genética , Locusta migratoria/metabolismo , Pupa/metabolismo , Interferência de RNA , Tribolium/genética , Tribolium/metabolismo
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