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1.
PLoS Genet ; 16(1): e1008363, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31929524

RESUMO

Prevention of desiccation is a constant challenge for terrestrial organisms. Land insects have an extracellular coat, the cuticle, that plays a major role in protection against exaggerated water loss. Here, we report that the ABC transporter Oskyddad (Osy)-a human ABCA12 paralog-contributes to the waterproof barrier function of the cuticle in the fruit fly Drosophila melanogaster. We show that the reduction or elimination of Osy function provokes rapid desiccation. Osy is also involved in defining the inward barrier against xenobiotics penetration. Consistently, the amounts of cuticular hydrocarbons that are involved in cuticle impermeability decrease markedly when Osy activity is reduced. GFP-tagged Osy localises to membrane nano-protrusions within the cuticle, likely pore canals. This suggests that Osy is mediating the transport of cuticular hydrocarbons (CHC) through the pore canals to the cuticle surface. The envelope, which is the outermost cuticle layer constituting the main barrier, is unaffected in osy mutant larvae. This contrasts with the function of Snu, another ABC transporter needed for the construction of the cuticular inward and outward barriers, that nevertheless is implicated in CHC deposition. Hence, Osy and Snu have overlapping and independent roles to establish cuticular resistance against transpiration and xenobiotic penetration. The osy deficient phenotype parallels the phenotype of Harlequin ichthyosis caused by mutations in the human abca12 gene. Thus, it seems that the cellular and molecular mechanisms of lipid barrier assembly in the skin are conserved during evolution.

2.
Pest Manag Sci ; 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31994314

RESUMO

BACKGROUND: Fatty alcohols are the precursors of sex pheromone components, wax esters and hydrocarbons in insects. Fatty acyl-CoA reductases (FARs) are important enzymes required for the reduction of fatty alcohol and thereby contribute to the production of cuticular hydrocarbon (CHC). RESULTS: Based on bioinformatics analyses we identified 17 FAR genes in the brown planthopper, Nilaparvata lugens. RNA interference against these genes demonstrated that ten NlFAR genes were essential for the survival of N. lugens. For instance, knockdown of NlFAR5, 6, 11 or 15 was lethal and caused a slender body shape, while the old cuticles of the respective animals remained attached to the abdomen or failed to split open from the nota. Knockdown of NlFAR9 resulted in a phenotype, with a smooth body surface and a decrease in CHC amounts. Similarly, CHC deficiency in N. lugens resulted in increased adhesion of water droplets and secreted honeydew to the insect surface and the inability of N. lugens to survive in paddy fields with varying humidity. Knockdown of NlFAR1, 4, 5, 6, 8, 9, 11 and 13 additionally resulted in female adult infertility. CONCLUSION: The present study illustrates the structural and functional differences of FAR family genes and provides potential targets for RNA interference-based rice planthopper management. © 2020 Society of Chemical Industry.

3.
Insect Sci ; 27(1): 14-21, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31246335

RESUMO

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas com Domínio T/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
4.
PeerJ ; 7: e7802, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31592352

RESUMO

An important role of the insect cuticle is to prevent wetting (i.e., permeation of water) and also to prevent penetration of potentially harmful substances. This barrier function mainly depends on the hydrophobic cuticle surface composed of lipids including cuticular hydrocarbons (CHCs). We investigated to what extent the cuticle inward barrier function depends on the genotype, comprising mitochondrial and nuclear genes in the fruit fly Drosophila melanogaster, and investigated the contribution of interactions between mitochondrial and nuclear genotypes (mito-nuclear interactions) on this function. In addition, we assessed the effects of nutrition and sex on the cuticle barrier function. Based on a dye penetration assay, we find that cuticle barrier function varies across three fly lines that were captured from geographically separated regions in three continents. Testing different combinations of mito-nuclear genotypes, we show that the inward barrier efficiency is modulated by the nuclear and mitochondrial genomes independently. We also find an interaction between diet and sex. Our findings provide new insights into the regulation of cuticle inward barrier function in nature.

5.
Insect Biochem Mol Biol ; 112: 103206, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31425850

RESUMO

Wings are an indispensable structure in many insects for their foraging, courtship, escape from predators, and migration. Cuticular proteins are major components of the insect cuticle and wings, but there is limited information on how cuticular proteins may play an essential role in wing morphogenesis. We identified a wing-specific cuticular protein, LmACP7, which belongs to the RR-2 subfamily of CPR chitin-binding proteins in the migratory locust. LmACP7 was initially produced in epidermal cells and subsequently migrated to the exocuticle at the pre-ecdysial stage in adult wings. Depletion of LmACP7 transcripts by RNA interference markedly reduced its protein amounts, which consequently led to abnormal wing morphogenesis. The deformed wings were curved, wrinkled, and failed to fully expand. We further demonstrated that the deformation was caused by both severe damage of the endocuticle and death of the epidermal cells in the wings. Based on these data, we propose that LmACP7 not only serves as an essential structural protein in the wing but is also required for the integrity of wing epithelial cells. LmACP7 contributes to production of the wing endocuticle and to the morphogenesis of functional wings in the migratory locust.


Assuntos
Proteínas de Insetos/genética , Locusta migratoria/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Quitina/metabolismo , Células Epidérmicas/metabolismo , Proteínas de Insetos/metabolismo , Locusta migratoria/crescimento & desenvolvimento , Metamorfose Biológica/genética , Morfogênese/genética , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Interferência de RNA , Asas de Animais/anormalidades
6.
Insect Biochem Mol Biol ; 110: 128-135, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31108167

RESUMO

The chitin modifying deacetylases (CDA) CDA1 and CDA2 have been reported to play partially redundant roles during insect cuticle formation and molting and tracheal morphogenesis in various insect species. In order to distinguish possible functional differences between these two enzymes, we analyzed their function during wing development in the fruit fly Drosophila melanogaster. In tissue-specific RNA interference experiments, we demonstrate that DmCDA1 (Serpentine, Serp) and DmCDA2 (Vermiform, Verm) have distinct functions during Drosophila adult wing cuticle differentiation. Chitosan staining revealed that Serp is the major enzyme responsible for chitin deacetylation during wing cuticle formation, while Verm does not seem to be needed for this process. Indeed, it is questionable whether Verm is a chitin deacetylase at all. Atomic force microscopy suggested that Serp and Verm have distinct roles in establishing the shape of nanoscale bumps at the wing surface. Moreover, our data indicate that Verm but not Serp is required for the laminar arrangement of chitin. Both enzymes participate in the establishment of the cuticular inward barrier against penetration of xenobiotics. Taken together, correct differentiation of the wing cuticle involves both Serp and Verm in parallel in largely non-overlapping functions.


Assuntos
Amidoidrolases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Asas de Animais/crescimento & desenvolvimento , Amidoidrolases/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Pupa/enzimologia , Pupa/genética , Pupa/crescimento & desenvolvimento , Interferência de RNA , Asas de Animais/enzimologia
7.
Adv Exp Med Biol ; 1142: 5-18, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31102240

RESUMO

Chitin is a linear polysaccharide of the amino sugar N-acetyl glucosamine. It is present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes and arthropods and fungi. Generally, it is an important component of protective or supportive extracellular matrices that cover the tissue that produces it or the whole body of the organism. Chitin fibres associate with each other adopting one of three possible crystalline organisations, i.e. α-, ß- or γ-chitin. Usually, chitin fibre bundles interact with chitin-binding proteins forming higher order structures. Chitin laminae, which are two-dimensional sheets of α-chitin crystals with antiparallel running chitin fibres in association with ß-folded proteins, are primary constituents of the arthropod cuticle and the fibrous extracellular matrix in sponges. A tri-dimensional composite material of proteins coacervates and ß-chitin constitute hard biomaterials such as the squid beak. The molecular composition of γ-chitin-based structures that contribute to the physical barrier found in insect cocoons is less well studied. In principle, chitin is a versatile extracellular polysaccharide that in association with proteins defines the mechanical properties of tissues and organisms.


Assuntos
Quitina/química , Matriz Extracelular/química , Insetos , Animais , Configuração de Carboidratos , Invertebrados , Proteínas
8.
Insect Biochem Mol Biol ; 108: 32-43, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30885803

RESUMO

The cuticular hydrocarbon (CHC) biosynthetic pathways branches off from the synthesis of fatty acids. Fatty acid elongases (ELOs) are enzymes catalyzing the synthesis of long-chain fatty acids and thereby contribute to the diversification of CHCs. Based on bioinformatics analyses we identified 20 ELO genes in the brown planthopper, Nilaparvata lugens. RNA interference against these genes demonstrated that 9 NlELO genes were essential for the survival of N. lugens nymphs and adults. Indeed, knockdown of NlELOs 1, 3, 4, 7, 8, 9, 10, 12 and 18 caused lethal phenotypes with a thin and wizened body and reduced lipids in the fat body. Surface analysis by scanning electron microscopy and CHC quantification indicated that knockdown of NlELOs 2, 3, 8 and 16 additionally resulted in a smooth body surface and a decrease in CHC amounts. Therefore, we speculate that long-chain CHCs are needed for CHC attachment to the cuticle surface. CHC deficiency, in turn, resulted in increased adhesion of water droplets and secreted honeydew to the animal surface and the inability of N. lugens to survive in paddy fields with varying humidity. Our present study provides an initial comprehensive analysis of ELO gene functions in an insect, and may serve to better understand the biology of CHCs.


Assuntos
/genética , Hemípteros/genética , Proteínas de Insetos/genética , Animais , Hemípteros/enzimologia , Hemípteros/metabolismo , Hidrocarbonetos/metabolismo , Proteínas de Insetos/metabolismo , Tegumento Comum , Metabolismo dos Lipídeos/genética , Família Multigênica , Interferência de RNA
9.
Sci Rep ; 9(1): 5374, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926832

RESUMO

The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered Drosophila cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the schlaff (slf) gene coding for a putative C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.

10.
Arch Insect Biochem Physiol ; 100(4): e21535, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30672604

RESUMO

Previously, we showed that inhibition of the activity of fatty acid desaturases (Desat) perturbs signalling of the developmental timing hormone ecdysone in the fruit fly Drosophila melanogaster. To understand the impact of this effect on cuticle differentiation, a process regulated by ecdysone, we analysed the cuticle of D. melanogaster larvae fed with the Desat inhibitor CA10556. In these larvae, the expression of most of the key cuticle genes is normal or slightly elevated at day one of CA10556 feeding. As an exception, expression of twdlM coding for a yet uncharacterised cuticle protein is completely suppressed. The cuticle of these larvae appears to be normal at the morphological level. However, these animals are sensitive to desiccation, a trait that according to our data, among others, may be associated with reduced TwdlM amounts. At day two of CA10556 feeding, expression of most of the cuticle genes tested including twdlM is suppressed. Expression of cpr47Eb coding for a chitin-binding protein is, by contrast, highly elevated suggesting that Cpr47Eb participates at a specific compensation program. Overall, the cuticle of these larvae is thinner than the cuticle of control larvae. Taken together, lipid desaturation is necessary for a coordinated deployment of a normal cuticle differentiation program.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Dessaturases/antagonistas & inibidores , Ácidos Graxos/metabolismo , Animais , Drosophila melanogaster/efeitos dos fármacos , Inibidores Enzimáticos/química , Ácidos Graxos/química , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Umidade , Muda/efeitos dos fármacos , Muda/fisiologia
11.
Insect Biochem Mol Biol ; 101: 124-130, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30196080

RESUMO

Chitinases, key enzymes involved in degradation of chitin, have been repeatedly shown to play an indispensable role during insect post-embryonic molting processes at stage transitions. However, how chitinases affect insect embryonic development remains to be analyzed. In this study, we investigated the role of chitinase 5-1 (LmCht5-1) during embryonic development of the hemimetabolous insect Locusta migratoria. LmCht5-1 transcript levels were high in pro-nymphs during late embryogenesis. The respective protein localized to both the pro-nymphal and, to a much lesser extent, the newly formed nymphal cuticle. After injection of double stranded RNA against LmCht5-1 into 8 days old embryos, LmCht5-1 transcripts were strongly reduced. Most of dsLmCht5-1-injected pro-nymphs failed to develop to first-instar nymphs and died at or before hatching. Histological analyzes showed that degradation of the pro-nymph cuticle was blocked in these animals. At the ultra-structural level, we found that LmCht5-1 was needed for the degradation of the lamellar procuticle, while the separation of the procuticle from the epicuticle and epidermis (apolysis) was independent of LmCht5-1 function. Taken together, our results indicate that LmCht5-1 and other yet unknown degrading enzymes act in parallel at distinct positions of the cuticle during molting of the pro-nymph to the first-instar nymph during locust embryogenesis.


Assuntos
Quitinases/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Locusta migratoria/enzimologia , Muda/genética , Animais , Quitina/química , Quitinases/antagonistas & inibidores , Quitinases/metabolismo , Embrião não Mamífero , Hidrólise , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Locusta migratoria/genética , Locusta migratoria/crescimento & desenvolvimento , Microinjeções , Ninfa/enzimologia , Ninfa/genética , Ninfa/crescimento & desenvolvimento , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Drug Discov Today ; 23(10): 1746-1760, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29890226

RESUMO

Solute carrier membrane transporters (SLCs) control cell exposure to small-molecule drugs, thereby contributing to drug efficacy and failure and/or adverse effects. Moreover, SLCs are genetically linked to various diseases. Hence, in-depth knowledge of SLC function is fundamental for a better understanding of disease pathophysiology and the drug development process. Given that the model organism Drosophila melanogaster (fruit fly) expresses SLCs, such as for the excretion of endogenous and toxic compounds by the hindgut and Malpighian tubules, equivalent to human intestine and kidney, this system appears to be a promising preclinical model to use to study human SLCs. Here, we systematically compare current knowledge of SLCs in Drosophila and humans and describe the Drosophila model as an innovative tool for drug development.


Assuntos
Drosophila melanogaster/metabolismo , Desenvolvimento de Medicamentos/métodos , Proteínas Carreadoras de Solutos/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Modelos Animais , Especificidade da Espécie
13.
Proc Natl Acad Sci U S A ; 115(20): 5175-5180, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712872

RESUMO

Cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins. Why does an insect encode so many different CP genes in the genome? In this study, we use comprehensive large-scale technologies to study the full complement of CPs (i.e., the CP-ome) of the brown planthopper (BPH), Nilaparvata lugens, a major rice plant pest. Eight CP families (CPR, CPF, TWDL, CPLCP, CPG, CPAP1, CPAP3, and CPAPn) including 140 proteins in BPH, in which CPAPn is a CP family that we discovered. The CPG family that was considered to be restricted to the Lepidoptera has also been identified in BPH. As reported here, CPLCP family members are characterized by three conserved sequence motifs. In addition, we identified a testis protein family with a peritrophin A domain that we named TPAP. We authenticated the real existence of 106 proteins among the 140 CPs. RNA interference (RNAi) experiments were conducted against 135 CP genes in early- and late-instar nymphs and newly emerged female adults, demonstrating that 32 CPs were essential for BPH normal development or egg production. Combined RNAi experiments suggested redundant and complementary functions of the large number of CPs. Transcriptomic data revealed that the CP genes were expressed in a tissue-specific manner, and there were four clusters of developmental expression patterns. This study gives a comprehensive understanding of the roles of CPs in an insect cuticle.


Assuntos
Hemípteros/genética , Proteínas de Insetos/genética , Família Multigênica , Interferência de RNA , Transcriptoma , Animais , Variação Genética , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo
14.
Dev Biol ; 437(2): 75-83, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29518377

RESUMO

Terminal differentiation of an organ is the last step in development that enables the organism to survive in the outside world after birth. Terminal differentiation of the insect tracheae that ends with filling the tubular network with gas is not fully understood at the tissue level. Here, we demonstrate that yet unidentified valves at the end of the tracheal system of the fruit fly Drosophila melanogaster embryo are important elements allowing terminal differentiation of this organ. Formation of these valves depends on the function of the zona pellucida protein Trynity (Tyn). The tracheae of tyn mutant embryos that lack these structures do not fill with gas. Additionally, external material penetrates into the tracheal tubes indicating that the tyn spiracles are permanently open. We conclude that the tracheal endings have to be closed to ensure gas-filling. We speculate that according to physical models closing of the tubular tracheal network provokes initial increase of the internal hydrostatic pressure necessary for gas generation through cavitation when the pressure is subsequently decreased.


Assuntos
Proteínas de Drosophila/metabolismo , Larva/metabolismo , Organogênese/fisiologia , Traqueia/embriologia , Animais , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero , Organogênese/genética , Traqueia/metabolismo
15.
Arch Insect Biochem Physiol ; 98(2): e21454, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29479741

RESUMO

This microreview stems from the Second Symposium on Insect Molecular Toxicology and Chitin Metabolism held at Shanxi University in Taiyuan, China (June 27 to 30, 2017) at the institute for Applied Biology headed by Professor Enbo Ma and Professor Jianzhen Zhang.


Assuntos
Quitina Sintase/metabolismo , Quitina/metabolismo , Insetos/metabolismo , Animais
16.
Eur J Cell Biol ; 97(2): 90-101, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29306642

RESUMO

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Pele/metabolismo , Animais , Drosophila , Matriz Extracelular/metabolismo , Metabolismo dos Lipídeos
17.
Insect Biochem Mol Biol ; 92: 1-11, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29113754

RESUMO

During growth and development of insects, the steroid hormone 20-Hydroxyecdysone (20E) regulates the molting process through activation of a series of genes including E74, E75 and HR3 by the 20E receptor EcR. Here, we analyzed the function of LmHR3 in the migratory locust Locusta migratoria. By sequence comparison, we first identified and characterized the putative nuclear receptor protein (LmHR3) based on L. migratoria transcriptome data. The full length cDNA is 2272 bp long encoding a protein of 455 amino acids that contains a DNA binding domain (zinc finger) and a ligand binding domain. Phylogenetic analyses showed that LmHR3 has a high homology with the ortholog from Blattaria. RT-qPCR results revealed that LmHR3 has a low level expression in the early days of 5th instar nymphs, and then increases and peaks at day 6, followed by a decrease to low levels before ecdysis. The LmHR3, hence, coincides with the profile of circulating 20E levels. Indeed, we show that transcription of LmHR3 is induced by 20E in vivo, and significantly suppressed by successfully knocking down expression of LmEcR. After injection of dsRNA for LmHR3 (dsLmHR3) at day 2 of earlier instar nymphs (3rd and 4th instar) and final instar nymphs (5th instar), none of the nymphs were able to molt normally, and eventually died. Chitin staining and ultra-structural analysis showed that both the synthesis of the new cuticle and the degradation of the old cuticle were blocked in the dsLmHR3 treated nymphs. Especially, chitin synthesis genes (LmUAP1 and LmCHS1) and chitinase genes (LmCHT5 and LmCHT10) were significantly down-regulated in the dsLmHR3 treatment group. Together, our results suggest that LmHR3 is involved in the control of chitin synthesis and degradation during L. migratoria molting.


Assuntos
Quitina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Locusta migratoria/genética , Muda/genética , Receptores Citoplasmáticos e Nucleares/genética , Sequência de Aminoácidos , Animais , Quitinases/genética , Quitinases/metabolismo , Ecdisterona/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Locusta migratoria/crescimento & desenvolvimento , Locusta migratoria/metabolismo , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Filogenia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência
18.
PLoS One ; 12(10): e0185897, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28977027

RESUMO

The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in Drosophila have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Drosophila/metabolismo , Endocitose , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Morfogênese , Transporte Proteico
19.
Insect Biochem Mol Biol ; 89: 1-10, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28821399

RESUMO

The insect cuticle is an extracellular matrix that consists of the polysaccharide chitin, proteins, lipids and organic molecules that are arranged in distinct horizontal layers. In Drosophila melanogaster, these layers are not formed sequentially, but, at least partially, at the same time. Timing of the underlying molecular mechanisms is conceivably crucial for cuticle formation. To study this issue, we determined the time period during which the function of Knickkopf (Knk), a key factor of chitin organization, is required for wing cuticle differentiation in D. melanogaster. Although knk is expressed throughout metamorphosis, we demonstrate that its expression 30 h prior and 48 h after pupariation is essential for correct wing cuticle formation. In other words, expression beyond this period is futile. Importantly, manipulation of Knk expression during this time causes wing bending suggesting an effect of Knk amounts on the physical properties of the wing cuticle. Manipulation of Knk expression also interferes with the structure and function of the cuticle surface. First, we show that the shape of surface nano-structures depends on the expression levels of knk. Second, we find that cuticle impermeability is compromised in wings with reduced knk expression. In summary, despite the extended supply of Knk during metamorphosis, controlled amounts of Knk are important for correct wing cuticle differentiation and function in a concise period of time.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Animais , Drosophila melanogaster/ultraestrutura , Feminino , Masculino , Melaninas/metabolismo , Metamorfose Biológica , Asas de Animais/metabolismo
20.
Insect Biochem Mol Biol ; 87: 90-99, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28610908

RESUMO

ATP-binding cassette (ABC) transporters constitute a large superfamily of proteins that mediate transport of a diverse number of substrates including nutrients, lipids and xenobiotics across membranes serving a variety of developmental and physiological functions. Here, we report on the molecular properties and biological roles of the ABC transporter LmABCH-9C in the migratory locust Locusta migratoria. LmABCH-9C was expressed continuously during nymphal development in all tissues including the integument. Expression was highest just after molting. Suppression of LmABCH-9C transcript levels by RNA interference (RNAi) in nymphs provoked death during or soon after molting to the next stage. These nymphs lost weight within minutes after molting. Moreover, high humidity rescued the lethality of molted dsLmABCH-9C-injected nymphs. In histological experiments, we find that the amounts of inner-cuticular lipids are reduced in nymphs with suppressed LmABCH-9C expression. These data together indicate that LmABCH-9C is needed for lipid-dependent desiccation resistance, paralleling the function of ABCH-9C in Tribolium castaneum. Hence, the function of this ABC transporter seems to be conserved across insect species ranging from hemimetabolous (L. migratoria) to holometabolous (T. castaneum) species. In addition, we find that cuticle inward impermeability is compromised in nymphs with reduced LmABCH-9C function. In summary, consistent with the model that cuticular lipids are necessary to prevent desiccation and penetration of xenobiotics in insects, we hypothesize that LmABCH-9C is involved in the construction of a lipid-based barrier at the surface of the cuticle especially after molting to protect the animal against uncontrolled water loss and entry. Susceptibility of this ABC transporter to RNAi-mediated knockdown designates it as an excellent target for RNAi-based insect pest control.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Insetos/genética , Locusta migratoria/crescimento & desenvolvimento , Muda/fisiologia , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Umidade , Proteínas de Insetos/deficiência , Proteínas de Insetos/metabolismo , Tegumento Comum/crescimento & desenvolvimento , Locusta migratoria/metabolismo , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Interferência de RNA
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