RESUMO
BACKGROUND: Diagnosis and immunotherapy of allergy against mites is based on complex extracts from large-scale cultures. However, the analysis of their composition using specific antibodies is limited. By taking advantage of the prevailing enzymatic nature of mite allergens, we have developed a broad-spectrum biochemical method for the standardization of native mite products. METHODS: Microplate-based assays have been implemented for thirteen Dermatophagoides pteronyssinus enzymatic activities, associated with Der p 1, 3, 4, 6, 8, 9, 15 and 20 allergens. The dynamics of these activities along culture growth, and their profile in purified fractions (bodies and faeces) and international reference standards (WHO/IUIS, two CBER/FDA), have been characterized. The stability of enzymatic activities and major allergens under stress conditions (40°C) has been assessed in the presence/absence of specific protease inhibitors. RESULTS: The analysis of enzymatic activities revealed distinct profiles along culture growth and between fractions (bodies and faeces). Remarkable differences were found when comparing international reference standards, being consistent with their source material (purified bodies or whole cultures). After 72 h at 40°C, only trypsin and alpha-amylase maintained high activity. Notably, the prominent role of trypsins in the hydrolytic degradation of major allergens is demonstrated by the use of inhibitors. CONCLUSIONS: Our method offers a robust approach to assess the complexity of mite extracts and highlights the critical importance of source materials for the composition and stability of finished products. The implementation of this approach in industry-based quality control procedures would contribute to the standardization of allergenic extracts used for diagnosis and immunotherapy.
Assuntos
Alérgenos/imunologia , Alérgenos/metabolismo , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/normas , Pyroglyphidae/enzimologia , Pyroglyphidae/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/normas , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/normas , Dermatophagoides pteronyssinus/enzimologia , Dermatophagoides pteronyssinus/imunologia , Ativação Enzimática , Estabilidade Enzimática , Humanos , Hipersensibilidade/diagnóstico , Controle de Qualidade , Padrões de Referência , Especificidade por SubstratoRESUMO
The expression of allergen genes in house dust mites is influenced by temperature and relative humidity, but little is known of the impacts of other environmental factors that may alter the repertoire of allergens released by mites in home microhabitats. Bioassays were conducted in concave microscope slides in combination with real-time quantitative polymerase chain reaction (RT-qPCR) to analyse gene expression of 17 allergens of Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) exposed to three chemical stressors that can be present in domestic environments. Short-term exposure (5-12 days) to diesel exhaust particles (DEPs) (1 µg/cm2 ), bacterial lipopolysaccharide endotoxin (0.1 µg/cm2 ) and benzyl benzoate (3.2 µg/cm2 ), at concentrations exceeding those expected in homes, had no significant effect on allergen transcription. A significant increase in the transcription of allergens Der p 3, Der p 8 and Der p 21 was observed only after exposing mites to a higher concentration of DEPs (10 µg/cm2 ) over a whole generation. In combination, the present results suggest that the analysed factors have low impact on allergen production. The methodology described here offers a sound and rapid approach to the broad-spectrum study of factors affecting allergen-related mite physiology, and allows the simultaneous screening of different factors in a relatively short period with consideration of the full spectrum of allergen genes.
Assuntos
Poluentes Atmosféricos/análise , Alérgenos/genética , Dermatophagoides pteronyssinus/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Inseticidas/análise , Lipopolissacarídeos/fisiologia , Alérgenos/metabolismo , Animais , Benzoatos/análise , Dermatophagoides pteronyssinus/genética , Dermatophagoides pteronyssinus/metabolismo , Material Particulado/análise , Emissões de Veículos/análiseRESUMO
Products manufactured from mass-cultured house dust mites, currently commercialized for the diagnosis and immunotherapy of allergy, are heterogeneous in terms of allergen composition and thus present concerns to regulatory authorities. The most abundant species, Dermatophagoides pteronyssinus (Trouessart) (Astigmata: Pyroglyphidae), produces 19 allergenic proteins. Many of these are putatively involved in mite digestive physiology and metabolism. This study aimed to evaluate the effects of mite-rearing media on allergen production. Mites were adapted to feed on culture media supplemented with proteins, lipids, carbohydrates or beard shavings, and collected to quantify major allergens (Der p 1 and 2) by immunodetection, transcription of allergen genes by real-time quantitative polymerase chain reaction, and allergen-related enzymatic activities. All culture media significantly affected the content of major allergens. Modification of macronutrients in the diet produced minor effects on the transcription of allergen genes, but significantly altered mite allergen-related activities. The most remarkable impacts were detected in mites feeding on beard shavings and were reflected in reductions in the content of major allergens, alterations in the transcription of nine allergen genes, and changes in eight allergen-related activities. These results demonstrate the importance of culture media to the quality and consistency of mite extracts used for pharmaceuticals, and highlight the need to further elucidate allergen production by mites in the laboratory and in domestic environments.
Assuntos
Alérgenos/metabolismo , Dermatophagoides pteronyssinus/fisiologia , Carboidratos da Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Lipídeos/administração & dosagem , Alérgenos/genética , Ração Animal/análise , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dermatophagoides pteronyssinus/enzimologia , Dermatophagoides pteronyssinus/genética , Dieta , Suplementos Nutricionais/análise , Expressão Gênica , PeleRESUMO
House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae) allergens (Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use.
Assuntos
Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Dermatophagoides pteronyssinus/genética , Regulação da Expressão Gênica , Animais , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Dermatophagoides pteronyssinus/crescimento & desenvolvimento , Dermatophagoides pteronyssinus/metabolismo , Meio Ambiente , Feminino , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species.
Assuntos
Dessensibilização Imunológica/métodos , Monitoramento Ambiental/métodos , Ácaros/classificação , Ácaros/genética , Alérgenos/imunologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 5,8S/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. Insecticide treatments for the control of this pest are mainly based on aerial and ground treatments with malathion bait sprays. However, the frequency of insecticide treatments has been increased in some areas of the Comunidad Valenciana in the last years, because of problems with the control of C. capitata. We have found that field populations from citrus and other fruit crops from different geographical areas in Spain showed lower susceptibility to malathion (6- to 201-fold) compared with laboratory populations. More importantly, differences in susceptibility could be related to the frequency of the field treatments. A resistant strain (W), derived from a field population, and a susceptible laboratory strain (C) were maintained in the laboratory. The W strain showed cross-resistance to the organophosphate fenthion (10-fold) but not to spinosad. Enzymatic assays showed that acethylcholinesterase activity was less inhibited in vivo by malathion and in vitro by malaoxon (active form of malathion) in adult flies from the W-resistant strain. Experiments to evaluate the effects of synergists revealed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) partially suppressed malathion resistance. Thus, target site insensitivity and metabolic resistance mediated by esterases might be involved in the loss of susceptibility to malathion in C. capitata. Nonetheless, additional biochemical and molecular studies will be required to confirm this hypothesis.
Assuntos
Ceratitis capitata/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Malation/farmacologia , Acetilcolinesterase , Animais , Demografia , Sinergistas de Praguicidas/farmacologia , EspanhaRESUMO
The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.
Assuntos
Variação Genética , Mariposas/enzimologia , Tripsina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Isoenzimas , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tripsina/genéticaRESUMO
Based on virion morphology, the current virus taxonomy groups entomopoxviruses (EPVs) (Poxvirus: Entomopoxvirinae) from coleopteran and dipteran hosts in separated genera, wilts it keeps viruses infecting either lepidopteran or orthopteran hosts in the same genus. In contrast to the morphological criteria, the few data available from recent studies at the genetic level have suggested that EPVs infecting different insect orders are phylogenetically distant. In order to elucidate EPVs phylogeny we have cloned and sequence the highly conserved/highly expressed spheroidin gene of Anacridium aegyptium entomopoxvirus (AaEPV). This gene and its promoter is of interest for the development of genetic engineering on EPVs. The spheroidin gene was located in the AaEPV genome by Southern blot and hybridisation with specific degenerated oligonucleotides probes synthesised after partial sequencing of the purified spheroidin protein. A total of 3489 bp were sequenced. This sequence included the coding and promoter region of 969 residues 108. 8 kDa protein identified as spheroidin. AaEPV spheroidin contains 21 cysteine residues (2.2%) and 14 N-glycosylation putative sites distributed along the sequence. The cysteine residues are particularly abundant at the C-terminal end of the protein, with 11 residues in the last 118 aa. Our results confirm that the spheroidin is highly conserved only between EPVs isolated from the same insect order. Polyclonal antibodies raised against AaEPV spherules specifically revealed spheroidin in Western Blots failing to cross-react with MmEPV or AmEPV spheroidins or MmEPV fusolin. Comparison of spheroidins at the aa level demonstrate that AaEPV spheroidin shares only 22.2 and 21.9% identity with the lepidopteran AmEPV and the coleopteran MmEPV spheroidins, respectively, but 82.8% identity with the orthopteran MsEPV spheroidin. Only two highly conserved domains containing the sequence (V/Y)NADTG(C/L) and LFAR(I/A) have been identified in all known spheroidins. The phylogenetic tree constructed according to the CLUSTALX analysis program revealed that EPVs are clearly separated in three groups - lepidopteran, coleopteran and orthopteran - according to the insect order of the virus hosts. In base to our results, the split of the genus Entomopoxvirus B dissociating lepidopteran and orthopteran EPVs into two different genera is suggested.
Assuntos
Entomopoxvirinae/genética , Genes de Insetos , Gafanhotos/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Besouros/virologia , Entomopoxvirinae/química , Lepidópteros/virologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Proteínas Virais/química , Proteínas Estruturais ViraisRESUMO
The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene.
Assuntos
Acetilcolinesterase/genética , Besouros/genética , Sequência de Aminoácidos , Animais , Inibidores da Colinesterase , Besouros/enzimologia , Besouros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Resistência a Inseticidas , Inseticidas , Dados de Sequência Molecular , Organofosfatos , Fenótipo , Interferência de RNA , Análise de Sequência de DNARESUMO
Cyclorrhapha insect genomes contain a single acetylcholinesterase (AChE) gene while other insects contain at least two ace genes (ace1 and ace2). In this study we tested the hypothesis that the two ace paralogous from Blattella germanica have different contributions to AChE activity, using RNA interference (RNAi) to knockdown each one individually. Paralogous-specific depletion of Bgace transcripts was evident in ganglia of injected cockroaches, although the effects at the protein level were less pronounced. Using spectrophotometric and zymogram measurements, we obtained evidence that BgAChE1 represents 65-75% of the total AChE activity in nerve tissue demonstrating that ace1 encodes a predominant AChE. A significant increase in sensitivity of Bgace1-interfered cockroaches was observed after 48 h of exposure to chlorpyrifos. In contrast, Bgace2 knockdown had a negligible effect on mortality to this organophosphate. These results point out a key role, qualitative and/or quantitative, of AChE1 as target of organophosphate insecticides in this species. Silencing the expression of Bgace1 but not Bgace2 also produced an increased mortality in insects when synergized with lambda-cyhalothrin, a situation which resembles the synergistic effects observed between organophosphates and pyrethroids. Gene silencing of ace genes by RNAi offers an exciting approach for examining a possible functional differentiation in ace paralogous.
Assuntos
Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Blattellidae/enzimologia , Inseticidas/farmacologia , Interferência de RNA , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Resistência a InseticidasRESUMO
The banana weevil (BW), Cosmopolites sordidus (Coleoptera: Curculionidae), is one of the most important insect pests of bananas and plantains. The mobility and the origin of BW infestations at the Canary Islands (Tenerife, La Gomera and La Palma) have been analysed using Random Amplified Polymorphic DNA (RAPD) as molecular markers. Populations from Costa Rica, Colombia, Uganda and Madeira were also included for comparison. One hundred and fifteen reproducible bands from eight primers were obtained. The level of polymorphism in the populations from the Canary Islands (40-62%) was in the range of those found in other populations. Nei's genetic distances, pair-wise fixation index (FST) values indicate that the closest populations are Tenerife populations among themselves (Nei's genetic distance=0.054-0.100; FST=0.091-0.157) and Costa Rica and Colombia populations (Nei's genetic distance=0.049; FST=0.113). Our results indicate the existence of BW local biotypes with limited gene flow and affected by genetic drift. These results are compatible with a unique event of colonization at Tenerife; whereas, the outbreaks in La Gomera and La Palma may come from independent introductions. The Madeira population is phylogenetically and geographically closer to the Canary Islands populations, suggesting that it is the most likely source of the insects introduced in the Canary Islands.
Assuntos
Variação Genética , Musa/parasitologia , Gorgulhos/genética , Animais , Ilhas Atlânticas , Genética Populacional , Interações Hospedeiro-Parasita , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.