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1.
Zool Res ; 42(5): 637-649, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472225

RESUMO

The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bombyx/crescimento & desenvolvimento , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Insetos/genética , Tegumento Comum/fisiologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Filogenia , Conformação Proteica
2.
Insect Sci ; 28(2): 533-547, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32166878

RESUMO

Spodoptera litura is a destructive agricultural pest in tropical and subtropical areas. Understanding the molecular mechanisms of S. litura adaptation to its preferred host plants may help identify target genes useful for pest control. We used high-throughput sequencing to characterize the expression patterns of messenger RNAs (mRNAs) and microRNAs (miRNAs) in the midgut of S. litura fed on Brassica juncea for 6 h and 48 h. A total of 108 known and 134 novel miRNAs were identified, 29 miRNAs and 237 mRNAs were differentially expressed at 6 h of B. juncea feeding, 26 miRNAs and 433 mRNAs were differentially expressed at 48 h. For the mRNAs, the up-regulated genes were mostly enriched in detoxification enzymes (cytochrome P450, esterase, glutathione S-transferase, uridine diphosphate-glucuronosyl transferase), while the down-regulated genes were mostly enriched in proteinases and immune-related genes. Furthermore, most detoxification enzymes begin to up-regulate at 6 h, while most digestion and immune-related genes begin to up- or down-regulate at 48 h. Eighteen and 37 differently expressed transcription factors were identified at 6 h and 48 h, which may regulate the functional genes. We acquired 136 and 41 miRNA versus mRNA pairs at 6 h and 48 h, respectively. Some down-regulated and up-regulated miRNAs were predicted to target detoxification enzymes and proteinases, respectively. Real-time quantitative polymerase chain reaction of nine randomly selected miRNAs and 28 genes confirmed the results of RNA-seq. This analyses of miRNA and mRNA transcriptomes provides useful information about the molecular mechanisms of S. litura response to B. juncea.


Assuntos
Herbivoria , MicroRNAs/análise , Mostardeira , Spodoptera/genética , Transcriptoma , Animais , Dieta , Trato Gastrointestinal , Larva/fisiologia
3.
Insect Sci ; 28(1): 47-62, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32283000

RESUMO

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.


Assuntos
Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Insetos/genética , Fatores de Transcrição Kruppel-Like/genética , Oócitos/crescimento & desenvolvimento , Transcriptoma , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo
4.
Insect Sci ; 27(6): 1257-1265, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31762161

RESUMO

The fall armyworm Spodoptera frugiperda recently invaded China, ravaging crops in many provinces. Deciphering the possible genetic basics for its successful invasion is critical for innovative and specific control for this gluttonous pest. Here we generated comparative genomic analyses between S. frugiperda and its native relative, S. litura, which differs in host preference, locomotivity and production behavior. We demonstrated that S. frugiperda genes are enriched in taste sensory perception and nervous system, obviously different from those of S. litura. Potential host adaptation genes showed generally an elevated ratio of non-synonymous substitution rate to synonymous substitution rate, suggesting a faster evolution during the divergence of the two species. Focusing on these sets of genes, we identified 23 genes being under positive selection in S. frugiperda. Among them are two notable genes involved in sensory perception, gustatory receptor (GR) and an acetaldehyde oxidase, which are important for host detection in invasion and expansion processes. Another two genes are mitochondrial adenosine triphosphate synthase ß subunit and ferritin heavy chain, which may be associated with the enhanced locomotivity and resistance, which fascinated long-distance migration needed for invasion and rapid expansion. Another interesting gene is chorion protein, in which positive selection sites in S. frugiperda were found and a replacement in one site is predicted to affect the protein function, which might be associated with competent reproductivity in S. frugiperda to ensure genetic resources for expansion.


Assuntos
Evolução Molecular , Genes de Insetos , Genoma , Spodoptera/fisiologia , Animais , China , Dieta , Espécies Introduzidas , Dinâmica Populacional , Seleção Genética , Spodoptera/genética
5.
Insect Sci ; 27(6): 1208-1223, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31840397

RESUMO

Metamorphosis is one of the most important physiological processes in insects. It is regulated by a serial of ecdysone cascade genes. Recently, lots of microRNAs (miRNAs) were investigated in insects; however, their function in metamorphosis is largely unknown. In the present study, the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis. A total of 101 miRNAs were identified, and 75 miRNAs were differentially expressed during the metamorphic process. The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs, and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets. Among them, miR-14-3p and its two targets (EcR and E75) were chosen for further validation. MiR-14-3p had higher expression level in the 6th instar larvae as compared with either the prepupae or pupae, which was opposite to that of both EcR and E75, two ecdysone cascade genes. Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p. Interestingly, the 3' untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes, including EcR, USP, E75, E74, E78, E93, Hr3, Hr4, Hr39, Krh1 and Ftzf1. Thus, different transcript variants of one ecdysone cascade gene could be regulated by the same miRNA. The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA. These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.


Assuntos
Ecdisona/metabolismo , Metamorfose Biológica/genética , MicroRNAs/genética , Transdução de Sinais/genética , Spodoptera/fisiologia , Animais , Sistema Digestório/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , MicroRNAs/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Spodoptera/genética , Spodoptera/crescimento & desenvolvimento , Transcriptoma
6.
Insect Sci ; 26(6): 1000-1010, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29808584

RESUMO

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/µL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.


Assuntos
Proteínas de Insetos/genética , Spodoptera/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Proteínas de Insetos/metabolismo , Masculino , Fenótipo , Spodoptera/crescimento & desenvolvimento , Spodoptera/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
7.
Insect Sci ; 26(5): 821-830, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29645353

RESUMO

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a "thin cocoon" mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like (Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the "thin cocoon" mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.


Assuntos
Bombyx/genética , Seda/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Fibroínas/genética , Proteínas de Insetos/genética , Larva/genética , Larva/metabolismo , Mutagênese Insercional/métodos , RNA Mensageiro , Seda/biossíntese
8.
Insect Sci ; 2017 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-29080335

RESUMO

Juvenile hormone (JH) is one of key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK cDNA was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and a pI of 4.55. Based on the structure three putative calcium binding motifs and GTP-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no ß-sheets. The gene was expressed in the epidermis, fat body and midgut of 5th and 6th instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of 5th and 6th instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura. This article is protected by copyright. All rights reserved.

9.
Insect Sci ; 23(6): 782-790, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25953667

RESUMO

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic "R&R" chitin-binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, "R&R" CBD peptide (CBD), non-CBD peptide (BmWCP4-CBD- ), four single site-directed mutated peptides (M1 , M2 , M3 and M4 ) and four-sites-mutated peptide (MF ) were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the "R&R" CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1 , M2 , M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.


Assuntos
Bombyx/genética , Quitina/metabolismo , Proteínas de Insetos/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
10.
Insect Sci ; 23(6): 819-828, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25959665

RESUMO

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no ß-sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Spodoptera/enzimologia , Motivos de Aminoácidos , Animais , Clonagem Molecular , Corpo Adiposo/enzimologia , Trato Gastrointestinal/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Juvenis/metabolismo , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Spodoptera/crescimento & desenvolvimento
11.
Insect Sci ; 23(5): 675-87, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25641855

RESUMO

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Glutathione S-transferases (GSTs) in insects are a family of detoxification enzymes and they play critical roles in CPF detoxification. Spodoptera litura is one of the most destructive agricultural pests in tropical and subtropical areas in the world. In this study, 37 Slgsts from 46 unique transcripts of gsts in S. litura transcriptome data, including eight previously reported GSTs, were identified and their expression patterns in susceptible and 12-generation-CPF-treated strains were analyzed to understand the roles of these Slgsts in sublethal doses of CPF tolerance. The results indicate that the members of the S. litura GST superfamily could be distinguished into three major groups: one group, including six cytosolic Slgsts (SlGSTe1, SlGSTe3, SlGSTe10, SlGSTe15, SlGSTo2 and SlGSTs5) and two microsomal Slgsts (SlMGST1-2 and SlMGST1-3), was directly responsible for CPF induction in both 12-generation-treated and susceptible strains; the second group, including three cytosolic Slgsts (SlGSTe13, SlGSTt1 and SlGSTz1) and one microsomal Slgst (SlMGST1-1), was induced only in the 12-generation-treated strain; the third group, including eight cytosolic Slgsts (two epsilon, three delta, one omega, one zeta and one unclassified Slgst), was expressed 1.52-5.15-fold higher in the 12-generation-treated strain than in the susceptible strain.


Assuntos
Clorpirifos/farmacologia , Glutationa Transferase/genética , Inseticidas/farmacologia , Spodoptera/enzimologia , Spodoptera/genética , Transcriptoma , Animais , Perfilação da Expressão Gênica , Inativação Metabólica , Resistência a Inseticidas , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Malondialdeído/análise , Spodoptera/crescimento & desenvolvimento
12.
Sci Rep ; 5: 12542, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26212173

RESUMO

The migratory locust displays a reversible, density-dependent transition between the two phases of gregaria and solitaria. This phenomenon is a typical kind of behavior plasticity. Here, we report that COP9 signalosome complex subunit 7A (CSN7A) is involved in the regulation of locust phase transition. Firstly, 90 proteins were identified to express differentially between the two phases by quantitative proteomic analysis. Gregaria revealed higher levels in proteins related to structure formation, melanism and energy metabolism, whereas solitaria had more abundant proteins related to digestion, absorption and chemical sensing. Subsequently, ten proteins including CSN7A were found to reveal differential mRNA expression profiles between the two phases. The CSN7A had higher mRNA level in the gregaria as compared with the solitaria, and the mRNA amount in the gregaria decreased remarkably during the 32 h-isolation. However, the mRNA level in the solitaria kept constant during the crowding rearing. Finally and importantly, RNA interference of CSN7A in gregaria resulted in obvious phase transition towards solitaria within 24 h. It suggests that CSN7A plays an essential role in the transition of gregaria towards solitaria in the migratory locust. To our knowledge, it's the first time to report the role of CSN in behavior plasticity of animals.


Assuntos
Migração Animal/fisiologia , Estágios do Ciclo de Vida/fisiologia , Locusta migratoria/crescimento & desenvolvimento , Locusta migratoria/metabolismo , Proteoma/metabolismo , Comportamento Social , Animais , Complexo do Signalossomo COP9 , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Complexos Multiproteicos , Peptídeo Hidrolases
13.
Insect Sci ; 22(1): 95-105, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24395766

RESUMO

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone (20E) induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.


Assuntos
Mariposas/fisiologia , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Ecdisterona/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos , Hormônios Juvenis/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/fisiologia , Dados de Sequência Molecular , Muda/fisiologia , Mariposas/genética , Isoformas de Proteínas/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Esteroides/genética , Fatores de Transcrição/genética
14.
Insect Sci ; 22(4): 503-11, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24863567

RESUMO

Insect glutathione S-transferases (GSTs) play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST SlGSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. SlGSTE1 was significantly up-regulated by chlorpyrifos and xanthotoxin in the midgut of S. litura. The recombinant SlGSTE1 had Vmax (reaction rate of the enzyme saturated with the substrate) and Km (michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme) values of 27.95 ± 0.88 µmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respectively, and Vmax and Km values of 22.96 ± 0.78 µmol/min/mg and 0.83 ± 0.106 mmol/L for 1-chloro-2,4-dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant SlGSTE1 possessed high binding activities to the insecticides chlorpyrifos, deltamethrin, malathion, phoxim and dichloro-diphenyl-trichloroethane (DDT). SlGSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that SlGSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. SlGSTE1 also showed high peroxidase activity. All the results together indicate that SlGSTE1 may play an important role in the gut of S. litura to protect the insect from the toxic effects of these compounds and heavy metals.


Assuntos
Glutationa Transferase/metabolismo , Inseticidas/metabolismo , Metais Pesados/metabolismo , Spodoptera/metabolismo , Animais , Trato Gastrointestinal/metabolismo , Inativação Metabólica , Larva/metabolismo , Metoxaleno/metabolismo , Estresse Oxidativo
15.
BMC Genomics ; 15: 820, 2014 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-25261999

RESUMO

BACKGROUND: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed. RESULTS: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated. CONCLUSIONS: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Perfilação da Expressão Gênica , Proteínas de Insetos/genética , Metamorfose Biológica , Asas de Animais/crescimento & desenvolvimento , Animais , Bombyx/anatomia & histologia , Quitina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Análise de Sequência de RNA , Transdução de Sinais , Asas de Animais/metabolismo
16.
J Insect Physiol ; 61: 34-41, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24406661

RESUMO

Heat shock protein (Hsp) and its cognate protein (Hsc) play important roles in helping insects survive extreme temperatures. However, high level of Hsp expression usually brings negative physiological effects on organisms. The mechanism of this trade-off is unclear. In this study, a lepidopteran insect, the common cutworm Spodoptera litura, was stressed at different temperatures, and the impact on both thermotolerance and fecundity was examined. The mRNA levels of four Hsp/Hscs (Hsp90, Hsc90, Hsp70 and Hsc70) and two ecdysone receptors (EcRs, EcRA and EcRB1) in different stresses and during the larval-pupal metamorphosis were determined. The results revealed that the pre-acclamation at mild stress increased the thermotolerance but decreased the egg production in adults. During the stress process, the mRNA levels of all the Hsp/Hsc and ecdysone receptor genes were significantly up-regulated. The two Hsp/Hsc70s and EcRs revealed consistent expression profiles with each other during the larval-pupal metamorphosis. Co-immunoprecipitation and Western blotting analysis indicated that Hsp/Hsc70 interacted with EcRs. RNAi of Hsc70 decreased the mRNA levels of two 20E-induced genes such as E74B and E75. Hsp70 transferred from the cytoplasm to nucleus in response to cold stress. These data together suggest that Hsp/Hsc70 might be involved in the regulation of 20E signaling, and the protein-protein interaction between Hsp/Hsc70 and EcRs probably act as a bridge mediating the trade-off between high thermotolerance and physiological defects.


Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Proteínas de Insetos/genética , Receptores de Esteroides/genética , Spodoptera/fisiologia , Animais , Western Blotting , Proteínas de Choque Térmico/metabolismo , Imunoprecipitação , Proteínas de Insetos/metabolismo , Larva/genética , Larva/fisiologia , Metamorfose Biológica , Pupa/genética , Pupa/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Esteroides/metabolismo , Reprodução , Spodoptera/genética , Spodoptera/crescimento & desenvolvimento
17.
Insect Sci ; 21(4): 449-58, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23955994

RESUMO

Insect midgut secretes a semi-permeable peritrophic membrane (PM), which plays important roles in protecting the midgut and helping with food digestion. The lepidopteran larvae produce type 1 PM, which is degraded when insects develop into the metamorphic stages. However, the underlying mechanism is unclear. In the present study, two peritrophin-like proteins (peritrophin-57 and 37) were identified from the midgut expression sequence tag library and transcriptome of the common cutworm, Spodoptera litura. The temporal and spatial expression patterns and responses to the induction of 20-hydroxyecdysone (20E) and starvation were examined by real-time quantitative polymerase chain reaction according to their common sequence region. The chitin-binding activity was also studied using a competitor, calcofluor. The open reading frames are 1 554 and 1 020 bp, respectively. They shared four highly conserved peritrophin-A domains and were expressed only in the midgut rather than in the other tissues, including fat body, epidermis, Malpighian tube and hemolymph. Their transcriptional expression could only be detected at the larval stages rather than in eggs, prepupae, pupae and adults. The purified protein of peritrophin-37 bound to chitin in a dose-dependent manner. These results indicate that the two proteins are peritrophins, the structural components of PM. In addition, the messenger RNA levels of the two peritrophins were significantly down-regulated by 20E injection, whereas feeding/starvation had no effect on the expression. These findings suggest that the increase of 20E titer may be an important factor which controls the degradation of PM during metamorphosis.


Assuntos
Quitina/genética , Proteínas de Insetos/metabolismo , Larva/genética , Metamorfose Biológica , Pupa/genética , Spodoptera/genética , Animais , Sequência de Bases , Quitina/metabolismo , Sistema Digestório/metabolismo , Ecdisterona/metabolismo , Etiquetas de Sequências Expressas , Biblioteca Gênica , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Spodoptera/crescimento & desenvolvimento , Spodoptera/metabolismo , Inanição , Transcriptoma
18.
Insect Biochem Mol Biol ; 43(9): 794-808, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23796435

RESUMO

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, ß-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and ß-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.


Assuntos
Epiderme/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/metabolismo , Pupa/metabolismo , Spodoptera/crescimento & desenvolvimento , Spodoptera/genética , Transcriptoma , Animais , Quitina/genética , Quitina/metabolismo , Epiderme/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Pupa/genética , Pupa/crescimento & desenvolvimento , Spodoptera/metabolismo
19.
PLoS Genet ; 9(2): e1003273, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23459255

RESUMO

Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL) is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone)-transcription factor (Relish)-target gene (cathepsin L) regulatory pathway.


Assuntos
Catepsina L , Ecdisona , Corpo Adiposo , Mariposas , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Catepsina L/genética , Catepsina L/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ecdisona/genética , Ecdisona/metabolismo , Corpo Adiposo/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/crescimento & desenvolvimento , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
PLoS One ; 7(3): e33621, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470457

RESUMO

BACKGROUND: Out of total 3,081 assembled expressed sequence tags (ESTs) sequences representing 6,815 high-quality ESTs identified in three cDNA libraries constructed with RNA isolated from the midgut of Spodoptera litura, 1,039 ESTs showed significant hits and 1,107 ESTs did not show significant hits in BLAST searches. It is of interest to clarify whether or not these ESTs that did not show hits function in S. Litura. RESULTS: Twenty "no-hit" ESTs containing at least one putative open reading frame were selected for further expression analysis. The results from northern blot analysis showed that six of the selected ESTs are expressed in the larval midgut of this insect at different levels, suggesting that these ESTs represent true mRNA products, whereas the other 14 ESTs could not be detected. Homologues of the four larval midgut-predominant genes (Slmg2, Slmg7, Slmg9 and Slmg17) were detected in the genomes of other lepidopteran insects but not in Drosophila melanogaster. A novel gene, Slmg7, is expressed at a high level specifically in the midgut during each of the larval stages. Slmg7 is a single copy gene and encodes a 143-amino acids protein. The SLMG7 protein was localized to the cytoplasm of Spli-221 cells. CONCLUSIONS: Six ESTs from the no hit list are transcribed into mRNA and are mainly expressed in the midgut of S. litura. Slmg7 is a novel gene that is localized to the cytoplasm.


Assuntos
Etiquetas de Sequências Expressas , Proteínas de Insetos/genética , Spodoptera/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Biologia Computacional , Trato Gastrointestinal/metabolismo , Biblioteca Gênica , Proteínas de Insetos/análise , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Spodoptera/crescimento & desenvolvimento
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