RESUMO
The effects of the beech apricot, Labramia bojeri A. de Candolle (Sapotales: Sapotaceae), seed aqueous extract on the larval development of the velvetbean moth, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was evaluated. The extract inhibited larval development, pupal weight, and survival and emergence of adults. Digestive proteolytic activity in larval midgut and feces extracts was determined. Larvae fed 10 g/L of the aqueous extract showed a significant reduction in trypsin activity (~64%), when compared with control larvae. Trypsin and chymotrypsin activities were also detected in fecal material in aqueous-extract-fed larvae, with about ~4.5 times more trypsin activity than the controls. The results from dietary utilization experiments with A. gemmatalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food and an increase in approximate digestibility and metabolic cost. The effect of the extract suggests the potential use of L. bojeri seeds to inhibit the development of A. gemmatalis via oral exposure. The L. bojeri extract can be an alternative to other methods of control.
Assuntos
Controle de Insetos , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sapotaceae/química , Animais , Inseticidas/química , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/crescimento & desenvolvimento , Mariposas/enzimologia , Mariposas/crescimento & desenvolvimento , Extratos Vegetais/química , Pupa/efeitos dos fármacos , Pupa/enzimologia , Pupa/crescimento & desenvolvimento , Sementes/química , Glycine max/crescimento & desenvolvimentoRESUMO
BACKGROUND: Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. METHODS: Ae. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC. RESULTS: The proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases. CONCLUSION: The preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression of trypsin-like isoforms among the preimaginal stages, suggesting that some of these enzymes are stage specific. Additionally, a comparison of the peptidase expression between larvae from eggs collected in the natural environment and larvae obtained from the eggs of female mosquitoes maintained in colonies for a long period of time demonstrated that the proteolytic profile is invariable under such conditions.
Assuntos
Aedes/embriologia , Aedes/enzimologia , Proteínas de Insetos/análise , Serina Proteases/análise , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas de Insetos/química , Larva/enzimologia , Peso Molecular , Proteólise , Pupa/enzimologia , Serina Proteases/química , Zigoto/enzimologiaRESUMO
BACKGROUND: The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. METHODS: The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 µM of the fluorogenic substrate Z-Phe-Arg-AMC. RESULTS: Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. CONCLUSION: The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the repertoire of proteolytic enzymes in preimaginal stages of the insect. Finally, differences in the trypsin-like serine peptidase profile of preimaginal stages were observed, suggesting that such enzymes exert specific functions during the different stages of the life cycle of the insect.
Assuntos
Culex/embriologia , Culex/enzimologia , Vetores de Doenças , Serina Proteases/análise , Animais , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Gelatina/metabolismo , Concentração de Íons de Hidrogênio , Larva/enzimologia , Óvulo/enzimologia , Pupa/enzimologia , TemperaturaRESUMO
Phosphorylation and dephosphorylation of protein tyrosine residues constitutes a major biochemical regulatory mechanism for the cell. We report a transient increase in the total tyrosine phosphorylation of the Aedes aegypti head during the first days after emergence from the pupal stage. This correlates with an initial reduction in total head protein tyrosine phosphatase (PTP) activity. Similarly, phosphotyrosine (pTyr)-containing bands are seen in extracts prepared from both male and female heads and are spread among a variety of structures including the antennae, proboscis and the maxillary palps combined with the proboscis. Also, mosquitoes treated with sodium orthovanadate, a classical PTP inhibitor, show reduced blood-feeding activity and higher head tyrosine phosphorylation levels. These results suggest that pTyr-mediated signalling pathways may play a role in the initial days following the emergence of the adult mosquito from the pupal stage.
Assuntos
Aedes/enzimologia , Proteínas Tirosina Fosfatases/análise , Aedes/crescimento & desenvolvimento , Animais , Feminino , Cabeça , Masculino , Proteínas Tirosina Fosfatases/metabolismo , Pupa/enzimologiaRESUMO
Phosphorylation and dephosphorylation of protein tyrosine residues constitutes a major biochemical regulatory mechanism for the cell. We report a transient increase in the total tyrosine phosphorylation of the Aedes aegypti head during the first days after emergence from the pupal stage. This correlates with an initial reduction in total head protein tyrosine phosphatase (PTP) activity. Similarly, phosphotyrosine (pTyr)-containing bands are seen in extracts prepared from both male and female heads and are spread among a variety of structures including the antennae, proboscis and the maxillary palps combined with the proboscis. Also, mosquitoes treated with sodium orthovanadate, a classical PTP inhibitor, show reduced blood-feeding activity and higher head tyrosine phosphorylation levels. These results suggest that pTyr-mediated signalling pathways may play a role in the initial days following the emergence of the adult mosquito from the pupal stage.
Assuntos
Animais , Feminino , Masculino , Aedes/enzimologia , Proteínas Tirosina Fosfatases , Aedes/crescimento & desenvolvimento , Cabeça , Proteínas Tirosina Fosfatases , Pupa/enzimologiaRESUMO
The present report shows a partial biochemical characterization and life cycle expression of N-ß-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-ß-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis.
Assuntos
Ceratitis capitata/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas de Insetos/metabolismo , Animais , Sequência de Bases , Encéfalo/enzimologia , Ceratitis capitata/crescimento & desenvolvimento , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Derme/enzimologia , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/enzimologia , Dados de Sequência Molecular , Fenótipo , Pupa/enzimologiaRESUMO
Species of the genus Culex Linnaeus have been incriminated as the main vectors of lymphatic filariases and are important vectors of arboviruses, including West Nile virus. Sequences corresponding to a fragment of 478 bp of the cytochrome c oxidase subunit I gene, which includes part of the barcode region, of 37 individuals of 17 species of genus Culex were generated to establish relationships among five subgenera, Culex, Phenacomyia, Melanoconion, Microculex, and Carrollia, and one species of the genus Lutzia that occurs in Brazil. Bayesian methods were employed for the phylogenetic analyses. Results of sequence comparisons showed that individuals identified as Culex dolosus, Culex mollis, and Culex imitator possess high intraspecific divergence (3.1, 2.3, and 3.5%, respectively) when using the Kimura two parameters model. These differences were associated either with distinct morphological characteristics of the male genitalia or larval and pupal stages, suggesting that these may represent species complexes. The Bayesian topology suggested that the genus and subgenus Culex are paraphyletic relative to Lutzia and Phenacomyia, respectively. The cytochrome c oxidase subunit I sequences may be a useful tool to both estimate phylogenetic relationships and identify morphologically similar species of the genus Culex.
Assuntos
Culex/enzimologia , Culex/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Insetos/metabolismo , Mitocôndrias/genética , Animais , Brasil , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Insetos/genética , Larva/enzimologia , Larva/genética , Masculino , Filogenia , Subunidades Proteicas , Pupa/enzimologia , Pupa/genéticaRESUMO
This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.
Assuntos
Aedes/enzimologia , Serina Endopeptidases/biossíntese , Aedes/metabolismo , Animais , Larva/enzimologia , Larva/metabolismo , Peso Molecular , Pepstatinas/farmacologia , Fenantrolinas/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Pupa/enzimologia , Pupa/metabolismo , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
In holometabolous insects, there is a complete body remodeling from larva to adult. We determined in Ceratitis capitata that the transition from pre-pupa to pupa, 40 to 48 h after puparium formation (h APF), is a key moment of metamorphosis; when salivary glands, intestine, fat body, and muscles are in different stages of cell death. At 44-46 h APF, muscles from segments 1-3 (thoracic region) appeared fully disintegrated, whereas posterior muscles just started death processes. To understand some of the biochemical events eventually involved in histolytic processes during early metamorphosis, two cysteine peptidases coined "Metamorphosis Associated Cysteine Peptidase" (MACP-I and MACP-II) were purified to homogeneity from 40-46-h APF insects. Both enzymes were inhibited by Ep-475, a specific inhibitor of papain-like cysteine-peptidases. MACP-I is a single chain protein with an apparent molecular mass of 80 kDa and includes several isoforms with pI values of pH 6.25-6.35, 6.7, and 7.2. The enzyme has an optimum pH of 5.0 and its pH stability ranges from pH 4.0 to 6.0. The molecular weight and N-terminal sequence suggest that MACP-I might be a novel enzyme. MACP-II is an acidic single chain protein with a pI of pH 5.85 and an apparent molecular mass of 30 kDa. The enzyme is labile with a maximum stability in the pH range of 4.0 to 6.0 and an optimum pH among 5.0 to 6.0. MAPCP-II characteristics suggest it is a cathepsin B-like enzyme.
Assuntos
Ceratitis capitata/enzimologia , Cisteína Endopeptidases/metabolismo , Metamorfose Biológica/fisiologia , Animais , Ceratitis capitata/fisiologia , Cisteína Endopeptidases/isolamento & purificação , Pupa/enzimologiaRESUMO
Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D. austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1-3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4-9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.
Assuntos
Drosophila/enzimologia , Drosophila/crescimento & desenvolvimento , Esterases/metabolismo , Fatores Etários , Animais , Eletroforese em Gel de Poliacrilamida , Esterases/análise , Larva/enzimologia , Pupa/enzimologia , Especificidade da EspécieRESUMO
The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using alpha- and beta-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as beta-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as alpha-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.
Assuntos
Drosophilidae/enzimologia , Esterases/química , Fatores Etários , Animais , Brasil , Drosophilidae/genética , Eletroforese em Gel de Poliacrilamida , Esterases/antagonistas & inibidores , Esterases/biossíntese , Esterases/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Larva/enzimologia , Larva/genética , Masculino , Pupa/enzimologia , Pupa/genética , Fatores SexuaisRESUMO
Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.
Assuntos
Ornitina Descarboxilase/metabolismo , Tephritidae/enzimologia , Tephritidae/crescimento & desenvolvimento , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Corpo Adiposo/metabolismo , Feminino , Guanosina Trifosfato/farmacologia , Cinética , Larva/enzimologia , Ovário/metabolismo , Óvulo/metabolismo , Pupa/enzimologiaRESUMO
Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.
Assuntos
Abelhas/enzimologia , Catecol Oxidase/biossíntese , Ecdisterona/fisiologia , Precursores Enzimáticos/biossíntese , Monofenol Mono-Oxigenase/metabolismo , Animais , Abelhas/crescimento & desenvolvimento , Ecdisterona/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemolinfa/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Pupa/enzimologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Azida Sódica/farmacologia , TemperaturaRESUMO
We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues
Assuntos
Animais , Feminino , Abelhas/enzimologia , Sistema Digestório , Fosfatase Ácida/metabolismo , Núcleo Celular/enzimologia , Ovário/enzimologia , Abelhas/ultraestrutura , Sistema Digestório , Larva/enzimologia , Larva/ultraestrutura , Microscopia Eletrônica , Ovário/ultraestrutura , Pupa/enzimologia , Pupa/ultraestruturaRESUMO
We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues.