Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.631
Filtrar
1.
PLoS Pathog ; 16(8): e1008697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776976

RESUMO

The diamondback moth, Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacterium Bacillus thuringiensis (Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal that P. xylostella requires combined mutations in both PxABCC2 and PxABCC3 to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations of PxABCC2 and PxABCC3 that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella, with a mutation (RA2) causing the mis-splicing of PxABCC2 and another mutation (RA3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed that RA2 and RA3 were tightly linked to Cry1Ac resistance. Introgression of RA2 and RA3 enabled a susceptible strain (G88) of P. xylostella to obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role of PxABCC2 and PxABCC3 in Cry1Ac resistance, frameshift mutations were introduced into PxABCC2 and PxABCC3 singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion of PxABCC2 or PxABCC3 alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance in P. xylostella by demonstrating mutations within both PxABCC2 and PxABCC3 genes are required for high-level Cry1Ac resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mariposas/química , Mariposas/genética , Mariposas/metabolismo , Mutação , Alinhamento de Sequência
2.
Proc Natl Acad Sci U S A ; 117(32): 19209-19220, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723826

RESUMO

Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called anhydrobiosis. The only known example of anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki Recent studies have led to a better understanding of the molecular mechanisms underlying anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome-transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.


Assuntos
Dípteros/metabolismo , Proteínas de Insetos/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Dessecação , Dípteros/química , Dípteros/genética , Secas , Glicogênio/genética , Glicogênio/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/química , Larva/genética , Larva/metabolismo , Metaboloma , Transcriptoma , Trealose/metabolismo , Água/metabolismo
3.
Nat Commun ; 11(1): 2911, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518308

RESUMO

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.


Assuntos
Difosfato de Adenosina/química , Culex/química , Mosquitos Vetores , Proteínas e Peptídeos Salivares/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Biologia Computacional/métodos , Cristalografia por Raios X , Eicosanoides/química , Comportamento Alimentar , Perfilação da Expressão Gênica , Hemostasia , Humanos , Proteínas de Insetos/química , Ligantes , Nucleotídeos/química , Agregação Plaquetária , Ligação Proteica , Domínios Proteicos , Saliva/química , Termodinâmica
4.
Arch Insect Biochem Physiol ; 105(1): e21720, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32557681

RESUMO

The large-conductance calcium-activated potassium channel (BKCa ) plays an important role in the regulation of insect neural circuits and locomotion, and thus is a potential target of insecticides. In this study, iberiotoxin, an inhibitor of BKCa , was found to prolong the anesthetic time of ethyl acetate on Plutella xylostella larvae. Therefore, the coding sequence of slowpoke gene coding the alpha subunit of BKCa was cloned to investigate the function of this channel in P. xylostella, and the gene expression profile in the developmental stages and tissues was also characterized. The total length of pxslo DNA was more than 19.9 kb, which harbored four alternative splicing sites (ASP-A, ASP-C, ASP-E, and ASP-G), and the coding sequence of pxslo with the highest frequency of splicing (GenBank ID: MN938456) was 3,405 base pair. The characterized PxSlo protein contained conserved domains previously identified in other insects. Quantitative reverse transcription-polymerase chain reaction analysis showed that pxslo was expressed in all the developmental stages of P. xylostella, with the highest level in adults. In the larval stage, pxslo was mainly expressed in the head and epidermis, while a limited protein was expressed in the midgut. In the adult stage, pxslo was highly expressed in the head, followed by in the ovarian tubule, and was not expressed in the testis or wings. These results suggest that BKCa plays an important physiological role in P. xylostella and provides useful information for the functional study and screening of BKCa inhibitors.


Assuntos
Proteínas de Insetos/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Mariposas/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Óvulo/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Alinhamento de Sequência
5.
J Insect Sci ; 20(3)2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32365175

RESUMO

Six candidate sHSP genes were identified from the Glyphodes pyloalis transcriptome. All sHSP genes included full-length open reading frames and shared high similarity with the sequences of other lepidopteran species. These sHSP genes encoded 175-191 amino acid residues, and the predicted proteins had a molecular weight from 19.5 to 21.8 kDa. All GpsHSPs were expressed at lower levels at larval stages. All GpsHSPs were expressed at higher levels at diapaused, prepupal, or pupal stages, suggesting that sHSPs may be involved in metamorphosis in G. pyloalis. In addition to the developmental stage, extreme temperatures can induce variations in the expression of sHSPs genes. All GpsHSPs were significantly upregulated in larvae following exposure to heat shock, except GpHSP21.4 which downregulated at 4 h following exposure to the cold shock treatment. Furthermore, Starvation influenced the expression patterns of GpsHSPs as a function of the duration of food deprivation. Four GpsHSPs increased their expression with time of starvation until reaching to the peak level at 6 d of starvation. Finally, parasitism by the endoparasitoid Aulacocentrum confusum He et van Achterberg (Hymenoptera: Braconidae)-induced fluctuations in the expression of all GpsHSPs, and the expression varied with time after parasitization. Our results from this study strongly suggest functional differentiation within the sHSPs subfamily in G. pyloalis. The present study would provide further insight into the roles of sHSPs in G. pyloalis and novel avenues for promoting integrated management of this pest.


Assuntos
Proteínas de Choque Térmico Pequenas/genética , Proteínas de Insetos/genética , Mariposas/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Filogenia , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Alinhamento de Sequência
6.
J Insect Sci ; 20(3)2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32396202

RESUMO

A large number of ecdysteroid-regulated 16 kDa proteins (ESR16s) of insects have been isolated and annotated in GenBank; however, knowledge on insect ESR16s remain limited. In the present study, we characterized an ecdysteroid-regulated 16 kDa protein gene isolated in Chinese oak silkworm, Antheraea pernyi Guérin-Méneville ('ApESR16' in the following), an important silk-producing and edible insect. The obtained cDNA sequence of ApESR16 is 1,049 bp, harboring an open reading frame of 441 bp that encodes a polypeptide of 146 amino acids. CD-search revealed that ApESR16 contains the putative cholesterol/lipid binding sites on conserved domain Npc2_like (Niemann-Pick type C-2) belonging to the MD-2-related lipid-recognition superfamily. Sequence comparison revealed that ApESR16 exhibits 51-57% identity to ESR16s of lepidopteran insects, 36-41% identity to ESR16 or NPC2a of nonlepidopteran insects, and 28-32% identity to NPC2a of vertebrates, indicating a high sequence divergence during the evolution of animals. Phylogenetic analysis found that the used sequences were divided into two groups corresponding to vertebrates and invertebrates, and the used insect sequences were also well clustered according to their families. The A. pernyi ESR16 mRNA is expressed during all four developmental stages and in all tested tissues. Injection of 20-hydroxyecdysone (20-E) into A. pernyi diapausing pupae triggering diapause termination induced upregulation of ESR16 mRNA compared to the diapausing pupae, with the highest expression level at day 2 in the ovaries but day 12 in the fat body. Our results suggested that ApESR16 might be a diapause-related gene and plays a vital role in the pupal diapause of A. pernyi.


Assuntos
Ecdisteroides/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Mariposas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Masculino , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Filogenia , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Alinhamento de Sequência
7.
Arch Insect Biochem Physiol ; 104(2): e21670, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32196735

RESUMO

Most immune effectors are inducible to microbial pathogen infection while some are already present to act as prophylactic immunity against as yet unseen infection. This study identified secretory phospholipase A2 (sPLA2 ) as a prophylactic factor in diamondback moth, Plutella xylostella. Western blotting using a polyclonal antibody raised against other lepidopteran sPLA2 reacted specifically with ∼25 kDa protein, which was present at approximately 0.4 mM in the plasma of naïve larvae. Interrogation of P. xylostella transcriptomes revealed an open-reading frame for sPLA2 (Px-sPLA2 ), exhibiting high homology with other Group III sPLA2 s. Px-sPLA2 was expressed in all developmental stages. In the larval stage, bacterial challenge induced its expression in hemocytes and fat body but not in gut or epidermis. RNA interference (RNAi) suppressed Px-sPLA2 messenger RNA level and sPLA2 activity in plasma. An inhibition zone assay showed that Px-sPLA2 exhibited antibacterial activities against different species, because specific RNAi knockdown impaired the activity. The RNAi treatment also suppressed the cellular immune response assessed by hemocyte nodule formation and humoral immune response assessed by antimicrobial peptide gene expression. Finally, benzylideneacetone (BZA, a specific sPLA2 inhibitor) treatment inhibited plasma sPLA2 activity of naive larvae in a dose-dependent manner. An addition of BZA significantly increased the bacterial virulence of an entomopathogen, Bacillus thuringiensis. These results suggest that Px-sPLA2 is an immune-associated factor of P. xylostella and its relatively high level of concentration in the plasma of naive larvae strongly suggests its role as a prophylactic factor in defending against pathogens at early infection stages.


Assuntos
Imunidade Celular , Imunidade Humoral , Proteínas de Insetos/genética , Mariposas/genética , Mariposas/imunologia , Fosfolipases A2 Secretórias/genética , Sequência de Aminoácidos , Animais , Eicosanoides , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Mariposas/crescimento & desenvolvimento , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/metabolismo , Filogenia , Alinhamento de Sequência
8.
World J Microbiol Biotechnol ; 36(4): 56, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32211973

RESUMO

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17 µg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus.


Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Xenorhabdus/metabolismo , Animais , Toxinas Bacterianas/genética , Sítios de Ligação , Cromatografia Líquida , Clonagem Molecular , Proteínas de Insetos/química , Mariposas/classificação , Mariposas/metabolismo , Ligação Proteica , Domínios Proteicos , Espectrometria de Massas em Tandem , Técnicas do Sistema de Duplo-Híbrido , Xenorhabdus/genética
9.
Insect Biochem Mol Biol ; 120: 103337, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109588

RESUMO

As the counterparts of noradrenaline and adrenaline in vertebrates, octopamine (OA) regulates multiple physiological and behavioral processes in invertebrate. OA mediates its effects via binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been reported in several insects. However, little work was documented in hemipteran insects. We cloned a ß-adrenergic-like OAR (NcOA2B2) from Nephotettix cincticeps. NcOA2B2 shares high similarity with members of the OA2B2 receptor class. Transcript level of NcOA2B2 varied in various tissues and was highly expressed in the leg. After heterologous expression in CHO-K1 cells, NcOA2B2 was dose-dependently activated by OA (EC50 = 2.56 nM) and tyramine (TA) (EC50 = 149 nM). Besides putative octopaminergic agonists, dopaminergic agonists and amitraz and DPMF potently activated NcOA2B2 in a dose-dependent manner. Receptor activity was blocked by potential antagonists and was most efficiently antagonized by asenapine. Phentolamine showed both antagonist and agonist effects on NcOA2B2. Our results offer the important information about molecular and pharmacological characterization of an OAR from N. cincticeps that will provide the basis for forthcoming studies on its roles in physiological processes and behaviors, and facilitate the design of novel insecticides for pest control.


Assuntos
Regulação da Expressão Gênica , Hemípteros/genética , Proteínas de Insetos/genética , Receptores de Amina Biogênica/genética , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Dopamina/metabolismo , Hemípteros/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Octopamina/metabolismo , Filogenia , Receptores de Amina Biogênica/química , Receptores de Amina Biogênica/metabolismo , Alinhamento de Sequência , Tiramina/metabolismo
10.
Gene ; 737: 144446, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32035241

RESUMO

The homeotic complex (Hox) gene Ultrabithorax (Ubx) plays pivotal roles in modifying specific morphological differences among the second (T2), the third thoracic (T3), and the first abdomen (A1) segment in several insects. Whether Ubx regulates wing dimorphism and other morphological traits in the delphacid family (order Hemiptera) remains elusive. In this study, we cloned a full-length Ubx ortholog (NlUbx) from the wing-dimorphic planthopper Nilaparvata lugens, and identified two NlUbx isoforms. RNA-interference (RNAi)-mediated silencing of NlUbx in short-winged BPH nymphs significantly induced the development of wing-like appendages from T3 wingbuds, and this effect is likely mediated by the insulin/insulin-like signaling pathway. RNAi knockdown of NlUbx in long-winged BPH nymphs led to a transformation from hindwings to forewings. Additionally, silencing of NlUbx not only dramatically changed the T3 morphology, but also led to jumping defect of T3 legs. First-instar nymphs derived from parental RNAi had an additional leg-like appendages on A1. These results suggest that Ubx plays a role in determining some morphological traits in delphacid planthoppers, and thus help in understanding evolution of morphological characteristics in arthropods.


Assuntos
Hemípteros/genética , Proteínas de Insetos/genética , Asas de Animais/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Feminino , Técnicas de Silenciamento de Genes , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/química , Masculino , Alinhamento de Sequência , Asas de Animais/crescimento & desenvolvimento
11.
Insect Biochem Mol Biol ; 119: 103326, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31968227

RESUMO

The antifungal activity of insect chitinase has rarely been studied. Here, we show that chitinase ChtIV, which is specifically expressed in the midgut of Asian corn borer (Ostrinia furnacalis), has antifungal activity toward phytopathogenic fungi. ChtIV exhibited high stability and mycelial hydrolytic activity in the extreme midgut environment, which has a pH of 10 and is rich in proteases. Hyper-N-glycosylation and reduced electrostatic interactions ensure the stability of ChtIV in the midgut. The structural characteristics of ChtIV are similar to two plant antifungal chitinases but distinct from an insect chitinase for cuticular chitin degradation in both the substrate-binding cleft and auxiliary binding motif. Since the phytopathogenic fungi are those that frequently invade corn, ChtIV may play a role in insect immune system and become a potential pesticide target. The crystal structures of ChtIV and its complexes with penta-N-acetylchitopentaose (a substrate) and allosamidin (an inhibitor) were obtained, which may facilitate rational design of ChtIV inhibitors as agrichemicals.


Assuntos
Antifúngicos/química , Quitinases/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Animais , Quitinases/química , Microbioma Gastrointestinal/efeitos dos fármacos , Proteínas de Insetos/química , Larva/química , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/química , Mariposas/crescimento & desenvolvimento
12.
J Agric Food Chem ; 68(6): 1731-1740, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31951399

RESUMO

Diamide insecticides targeting ryanodine receptors (RyRs) are a major class of pesticides used to control a wide range of agricultural pests, but their efficacies have been reduced dramatically by the recent emergence of resistance mutations. There is a pressing need to develop novel insecticides, targeting distinct and novel binding sites within insect RyRs to overcome the resistance crisis; however, the limited structural information on insect RyRs is a major roadblock to our understanding of their molecular mechanisms. Here, we report the crystal structure of the RyR SPRY2 domain from the diamondback moth (DBM), Plutella xylostella, a destructive agricultural pest worldwide that has developed resistance to all classes of insecticide at 2.06 Å resolution. The overall fold of DBM SPRY2 is similar to its mammalian homolog, but it shows distinct conformations in several loops. Docking it into the recently published cryo-electron microscope structure of the full-length RyR reveals that two insect-specific loops interact with the BSol domain from the neighboring subunit. The SPRY2-BSol interface will change the conformation upon channel gating, indicating that it might be a potential targeting site for insect-specific insecticides. Interestingly, several previously identified disease-causing mutations also lie in the same interface, implying that this interface is important for channel gating. Another insect-specific loop located in the SPRY2-SPRY3 interface might indirectly affect another gating interface between SPRY3 and Repeat34.


Assuntos
Diamida/química , Proteínas de Insetos/química , Inseticidas/química , Mariposas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Diamida/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Simulação de Acoplamento Molecular , Mariposas/química , Mariposas/efeitos dos fármacos , Mariposas/genética , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
13.
Arch Insect Biochem Physiol ; 103(4): e21651, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31943343

RESUMO

DNA methylation refers to the addition of cytosine residues in a CpG context (5'-cytosine-phosphate-guanine-3'). As one of the most common mechanisms of epigenetic modification, it plays a crucial role in regulating gene expression and in a diverse range of biological processes across all multicellular organisms. The relationship between temperature and DNA methylation and how it acts on the adaptability of migratory insects remain unknown. In the present work, a 5,496 bp full-length complementary DNA encoding 1,436 amino acids (named MsDnmt1) was cloned from the devastating migratory pest oriental armyworm, Mythimna separata Walker. The protein shares 36.8-84.4% identity with other insect Dnmt1 isoforms. Spatial and temporal expression analysis revealed that MsDnmt1 was highly expressed in adult stages and head tissue. The changing temperature decreased the expression of MsDnmt1 in both high and low temperature condition. Besides, we found that M. separata exhibited the shortest duration time from the last instar to pupae under 36°C environment when injected with DNA methylation inhibitor. Therefore, our data highlight a potential role for DNA methylation in thermal resistance, which help us to understand the biological role adaptability and colonization of migratory pest in various environments.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas de Insetos/genética , Mariposas/fisiologia , Sequência de Aminoácidos , Animais , Temperatura Corporal , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Óvulo/crescimento & desenvolvimento , Óvulo/fisiologia , Filogenia , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Alinhamento de Sequência
14.
J Agric Food Chem ; 68(4): 982-988, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31909997

RESUMO

Cycloxaprid (CYC) is effective in the control of hemipteran pests, but its bioactivity against lepidopteran pests is still unclear. Here, the bioactivity of CYC against lepidopteran pests was found to be much worse than that against hemipteran insects. To reveal the mechanism, the transcriptomes of CYC-treated and untreated Ostrinia furnacalis larvae were compared. Among the top 20 differentially expressed genes, 11 encode proteins involved in cuticle formation, while only one encodes a detoxifying enzyme. Thus, the cuticle appears to be important for the insensitivity of O. furnacalis to CYC. A pretreatment of O. furnacalis larvae with methoprene enhanced the bioactivity of CYC by 1.12-fold. Moreover, mixtures of CYC with graphene oxide increased the bioactivity of CYC by 1.88-fold. Because lepidopteran and hemipteran insects often harm crops at the same time, the work can help make full use of CYC and reduce the environmental impacts of using multiple pesticides.


Assuntos
Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inseticidas/química , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Estrutura Molecular , Mariposas/química , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Alinhamento de Sequência
15.
Mol Phylogenet Evol ; 145: 106736, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31978488

RESUMO

Lamiinae is the most diverse subfamily of longhorned beetles, with about 20,000 described species classified into 80 tribes. Most of the tribes of Lamiinae were proposed during the 19th century and the suprageneric classification of the subfamily has never been assessed under phylogenetic criteria. In this study, we present the first tribal-level phylogeny of Lamiinae, inferred from 130 terminals (representing 46 tribes, prioritizing generic type species of the tribes) and fragments of two mitochondrial and three nuclear markers (cox1, rrnL, Wg, CPS and LSU; 5,024 aligned positions in total). Analyses were performed under Maximum Likelihood and Bayesian methods based on two datasets: a dataset including all taxa available for the study, and a reduced dataset with 111 terminals where taxa only contributing with mitochondrial markers were excluded from the matrix. The monophyly of Lamiinae was corroborated in three of the four analyses and 11 of the 35 tribes with more than one species represented in the analyses were consistently recovered as monophyletic. However, 15 tribes were not retrieved as monophyletic, requiring a revision of their boundaries: Acanthocinini, Acanthoderini, Agapanthiini, Apomecynini, Desmiphorini, Dorcaschematini, Enicodini, Hemilophini, Monochamini, Onciderini, Parmenini, Phytoeciini, Pogonocherini, Pteropliini and Saperdini. Based on these results, when strong support values for paraphyly were recovered, we argue a number of tribe synonymies, including Moneilemini as synonym of Acanthocinini; Onocephalini of Onciderini; Dorcadionini, Gnomini, Monochamini and Rhodopinini of Lamiini; and Obereini and Phytoeciini of Saperdini. Other taxonomic changes proposed in this study based on the criterion of monophyly and supported by morphological characters include the transfer of Tricondyloides and Stenellipsis to Enicodini, and of Dylobolus stat. rest., which is removed as subgenus of Mecas and restituted as genus, to Hemilophini. Furthermore, our analyses suggest that Ostedes and Neohoplonotus should be removed from Acanthocinini and Parmenini, respectively, and Colobotheini should be redefined to encompass several genera currently placed in Acanthocinini.


Assuntos
Besouros/classificação , Animais , Teorema de Bayes , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Funções Verossimilhança , Mitocôndrias/genética , Filogenia , Subunidades Ribossômicas Maiores/genética
16.
Anal Bioanal Chem ; 412(6): 1431-1439, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31912183

RESUMO

Triatominae are hematophagous insects involved in the transmission of Chagas disease. Among the 19 genera of the subfamily, those with the highest epidemiological importance regarding the dissemination of Trypanosoma cruzi are Panstrongylus, Rhodnius, and Triatoma. Of these three genera, Rhodnius presents the greatest difficulties for specific identification. Thus, there is a need to overcome the difficulties in identifying phenotypes of similar species of this genus. In the present study, the MALDI-TOF MS methodology was used to identify 12 Rhodnius species, among the 21 admitted. The MALDI-TOF MS methodology allowed specific characterization through the identification of peptides and proteins, starting from four different methods of extraction: (A) acetonitrile/formic acid (ACN/AF), (B) acetonitrile/trifluoroacetic acid (ACN/TFA), (C) isopropyl/formic acid (IPA/AF), and (D) methanol/formic acid (MeOH/AF), and four types of MALDI-TOF matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 6-aza-2-thiothymine (ATT), and 2,6-dihydroxyacetophenone (DHAP). The experiments were performed by combining the four solvents and four matrices to select the best MALDI extraction/matrix. The application of the MALDI-TOF MS technique, through the digital mass spectrometry approach combined with chemometric tools, such as partial least squares-discriminant analysis (PLS-DA), was able to discriminate 12 species of Rhodnius genus, which are difficult to identify using morphological characteristics. Thus, in view of the results obtained, the methodology described in the present article can be applied with speed and efficiency for the discrimination of Triatominae species. Graphical Abstract.


Assuntos
Proteínas de Insetos/química , Peptídeos/química , Rhodnius/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetofenonas/química , Animais , Ácidos Cumáricos/química , Formiatos/química
17.
PLoS One ; 15(1): e0225672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923175

RESUMO

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.


Assuntos
Hialuronoglucosaminidase/metabolismo , Proteínas de Insetos/metabolismo , Fosfolipases/metabolismo , Venenos de Vespas/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/isolamento & purificação , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Nanotecnologia , Fosfolipases/química , Fosfolipases/isolamento & purificação , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Venenos de Vespas/química , Venenos de Vespas/isolamento & purificação , Venenos de Vespas/metabolismo , Vespas
18.
Nature ; 578(7794): 311-316, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996847

RESUMO

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.


Assuntos
Motivos de Aminoácidos , Sequência Consenso , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Fosfolipase D/química , Fosfolipase D/metabolismo , RNA Interferente Pequeno/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Sistema Livre de Células , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Metilação , Camundongos , RNA Helicases/metabolismo
19.
Insect Mol Biol ; 29(1): 104-111, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31390480

RESUMO

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin-III (BmApoLp-III). First, the expression of BmApoLp-III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp-III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp-III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp-III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp-III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition.


Assuntos
Apolipoproteínas/química , Bombyx/química , Proteínas de Insetos/química , Estabilidade Proteica/efeitos dos fármacos , Acetilação , Animais , Apolipoproteínas/metabolismo , Benzoatos/farmacologia , Bombyx/efeitos dos fármacos , Linhagem Celular , Cicloeximida/farmacologia , Proteínas de Insetos/metabolismo , Leupeptinas/farmacologia , Panobinostat/farmacologia , Pirazóis/farmacologia
20.
Insect Biochem Mol Biol ; 117: 103290, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31790798

RESUMO

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.


Assuntos
Expressão Gênica , Proteínas de Insetos/genética , Lipopolissacarídeos/metabolismo , Manduca/genética , Receptores de Prostaglandina E Subtipo EP2/genética , Ácidos Teicoicos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica , Hemócitos/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Manduca/metabolismo , Filogenia , Receptores de Prostaglandina E Subtipo EP2/química , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Alinhamento de Sequência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA