RESUMO
The yeast-encapsulated orange oil (YEOO) is a novel larvicide under development against vector mosquitoes. Despite its efficiency against Aedes aegypti (L.) in small scale experiments, its applicability in vector control can be influenced by other effects on mosquito behaviour or physiology. For this reason, the impact of YEOO particles in mosquito oviposition was evaluated in laboratory and semi-field conditions. Oviposition assays with one gravid Aedes aegypti female were carried under laboratory and semi-field conditions with natural light and temperature fluctuation. For all ovitraps, the number of eggs was manually counted in the wooden paddle and in the solution of each ovitrap. The proportion of eggs between substrates (wooden paddle and solution) varied between conditions, with females in laboratory presenting a lower preference to lay eggs in paddles when compared with studies in semi-field. This behaviour shifts in laboratory can create challenges to extrapolate results from laboratory to the field. Here, studies in both conditions indicate a similar impact of YEOO particles in Aedes aegypti oviposition. The potential treatment concentration of YEOO particles presents a strong repellent/deterrent effect (-0.559 > OAI > -0.760) within the initial 72h of application when compared with water, and weak repellent/deterrent signal (OAI = -0.220) when compared against inactivated yeast. Control ovitraps with water were more positive for egg presence than treated ovitraps, while ovitraps with YEOO particles and inactivated yeast present similar number of positive ovitraps. It is possible that the repellent/deterrent action is partially driven by the delivery system, since most times Citrus sinensis EO oviposition repellent/deterrent signal is weak, and it seem influenced by solvent/delivery used. However, it is unclear how the yeast wall that protect/surrounds the orange oil will negatively affect oviposition since live yeast are normally consider an attractant for mosquito oviposition.
Assuntos
Aedes , Controle de Mosquitos , Oviposição , Óleos de Plantas , Aedes/fisiologia , Aedes/efeitos dos fármacos , Animais , Oviposição/efeitos dos fármacos , Feminino , Óleos de Plantas/farmacologia , Controle de Mosquitos/métodos , Mosquitos Vetores/fisiologia , Mosquitos Vetores/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Repelentes de Insetos/farmacologiaRESUMO
BACKGROUND: The resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain. METHODS: The activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined. RESULTS: Enzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters. CONCLUSIONS: Our dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation.
Assuntos
Bacillus , Toxinas Bacterianas , Culex , Animais , Feminino , Toxinas Bacterianas/metabolismo , Culex/metabolismo , Lipídeos , Larva/genéticaRESUMO
Chagas disease is a human infectious disease caused by Trypanosoma cruzi and can be transmitted by triatomine vectors, such as Rhodnius prolixus. One limiting factor for T. cruzi development is the composition of the bacterial gut microbiota in the triatomine. Herein, we analyzed the humoral immune responses of R. prolixus nymphs treated with antibiotics and subsequently recolonized with either Serratia marcescens or Rhodococcus rhodnii. The treatment with antibiotics reduced the bacterial load in the digestive tract, and the recolonization with each bacterium was successfully detected seven days after treatment. The antibiotic-treated insects, recolonized with S. marcescens, presented reduced antibacterial activity against Staphylococcus aureus and phenoloxidase activity in hemolymph, and lower nitric oxide synthase (NOS) and higher defensin C gene (DefC) gene expression in the fat body. These insects also presented a higher expression of DefC, lower prolixicin (Prol), and lower NOS levels in the anterior midgut. However, the antibiotic-treated insects recolonized with R. rhodnii had increased antibacterial activity against Escherichia coli and lower activity against S. aureus, higher phenoloxidase activity in hemolymph, and lower NOS expression in the fat body. In the anterior midgut, these insects presented higher NOS, defensin A (DefA) and DefC expression, and lower Prol expression. The R. prolixus immune modulation by these two bacteria was observed not only in the midgut, but also systemically in the fat body, and may be crucial for the development and transmission of the parasites Trypanosoma cruzi and Trypanosoma rangeli.
Assuntos
Antibacterianos/uso terapêutico , Rhodnius/microbiologia , Rhodococcus/imunologia , Serratia marcescens/imunologia , Animais , Antibacterianos/farmacologia , Defensinas/metabolismo , Corpo Adiposo/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Humoral , Proteínas de Insetos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Óxido Nítrico Sintase/metabolismo , Rhodnius/efeitos dos fármacos , Rhodnius/imunologia , Rhodnius/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
Beta-1,3-glucanases are enzymes that hydrolyze beta-1,3-glucans, and they are essential for the metabolism of seaweed, plants and fungi. These enzymes also participate in the digestion of herbivore and fungivore animals. Because of the importance of these enzymes in insects, beta-1,3-glucanase inhibitors may be used for the development of new control strategies against agricultural pests and disease vectors. Beta-1,3-glucanase inhibitors have been described in the brown seaweed Laminaria cichorioides, but were never recorded in Brazilian seaweed species. We evaluated the presence of beta-1,3-glucanase inhibitors in samples of Padina gymnospora, Dictyota sp., Colpomenia sinuosa, and Lobophora sp., collected in Arraial d'Ajuda (Bahia). Ethanolic or buffer extracts were used in inhibition tests against the beta-1,3-glucanase of Trichoderma sp. Extracts in buffer showed no inhibition, but ethanolic extracts from all species showed different extents of inhibition. Samples from Dictyota sp. and P. gymnospora showed inhibitions above 75% (absolute ethanol) or 50% (ethanol 50%). In summary, extraction with absolute ethanol resulted in better inhibitions, and P. gymnospora showed the higher inhibitions. Brazilian seaweed may be good sources of beta-1,3-glucanase inhibitors for biochemical and physiological studies of these enzymes. Besides that, these molecules show potential for the development of new biotechnological tools for insect control.
Assuntos
Alga Marinha , Animais , Brasil , Fungos , VerdurasRESUMO
BACKGROUND: Culex quinquefasciatus resistance to the binary toxin from Lysinibacillus sphaericus larvicides can occur because of mutations in the cqm1 gene that prevents the expression of the toxin receptor, Cqm1 α-glucosidase. In a resistant laboratory-selected colony maintained for more than 250 generations, cqm1REC and cqm1REC-2 resistance alleles were identified. The major allele initially found, cqm1REC , became minor and was replaced by cqm1REC-2 . This study aimed to investigate the features associated with homozygous larvae for each allele to understand the reasons for the allele replacement and to generate knowledge on resistance to microbial larvicides. RESULTS: Homozygous larvae for each allele were compared. Both larvae displayed the same level of resistance to the binary toxin (3500-fold); therefore, a change in phenotype was not the reason for the replacement observed. The lack of Cqm1 expression did not reduce the total specific α-glucosidase activity for homozygous cqm1REC and cqm1REC-2 larvae, which were statistically similar to the susceptible strain, using artificial or natural substrates. The expression of eight Cqm1 paralog α-glucosidases was demonstrated in resistant and susceptible larvae. Bioassays in which cqm1REC or cqm1REC-2 homozygous larvae were reared under stressful conditions showed that most adults produced were cqm1REC-2 homozygous (69%). Comparatively, in the offspring of a heterozygous sub-colony reared under optimal conditions for 20 generations, the cqm1REC allele assumed a higher frequency (0.72). CONCLUSION: Homozygous larvae for each allele exhibited a similar resistant phenotype. However, they presented specific advantages that might favor their selection and can be used in designing resistance management practices. © 2021 Society of Chemical Industry.
Assuntos
Toxinas Bacterianas , Culex , Proteínas de Insetos/genética , alfa-Glucosidases/genética , Alelos , Animais , Bacillaceae , Culex/enzimologia , Culex/genética , Larva/genéticaRESUMO
Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.
Assuntos
Catepsina B/genética , Catepsina L/genética , Hemípteros/fisiologia , Proteínas de Insetos/genética , Sequência de Aminoácidos , Fenômenos Fisiológicos da Nutrição Animal , Animais , Sequência de Bases , Catepsina B/química , Catepsina B/metabolismo , Catepsina L/química , Catepsina L/metabolismo , Digestão , Hemípteros/enzimologia , Hemípteros/genética , Heterópteros/enzimologia , Heterópteros/genética , Heterópteros/fisiologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Rhodnius/enzimologia , Rhodnius/genética , Rhodnius/fisiologiaRESUMO
Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.
RESUMO
Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.
RESUMO
The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves ß-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive ß-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.
RESUMO
Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.
Assuntos
Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Triatoma/genética , Sequência de Aminoácidos , Animais , Carboxipeptidases/química , Catepsina A/química , Catepsina A/genética , Catepsina A/metabolismo , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Distribuição Tecidual , Triatoma/metabolismoRESUMO
The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop.
Assuntos
Tenebrio/enzimologia , Trealase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , Evolução Molecular , Trato Gastrointestinal/enzimologia , Glucosídeos/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Spodoptera/enzimologia , Trealase/antagonistas & inibidores , Trealase/isolamento & purificaçãoRESUMO
We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and ß-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a ß-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.
Assuntos
Amilases/metabolismo , Ensaios Enzimáticos/métodos , Glucana 1,3-beta-Glucosidase/metabolismo , Miniaturização/instrumentação , Ensaios Enzimáticos/instrumentação , Humanos , Cinética , Quinolinas/química , Salicilatos/química , Saliva/enzimologia , Amido/metabolismoRESUMO
Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.
Assuntos
Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Psychodidae/metabolismo , Animais , Trato Gastrointestinal/enzimologia , Glicólise , Larva/metabolismo , Psychodidae/enzimologia , Trealase/metabolismo , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismoRESUMO
Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.
Assuntos
Imunidade/imunologia , Leishmania mexicana/imunologia , Psychodidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Serratia marcescens/imunologia , Sequência de Aminoácidos , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Catalase/classificação , Catalase/genética , Catalase/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/parasitologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade/efeitos dos fármacos , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/imunologia , Insetos Vetores/microbiologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Dados de Sequência Molecular , Peroxirredoxinas/classificação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Psychodidae/enzimologia , Psychodidae/genética , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Serratia marcescens/fisiologia , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologiaRESUMO
BACKGROUND: The description of new hydrolytic enzymes is an important step in the development of techniques which use lignocellulosic materials as a starting point for fuel production. Sugarcane bagasse, which is subjected to pre-treatment, hydrolysis and fermentation for the production of ethanol in several test refineries, is the most promising source of raw material for the production of second generation renewable fuels in Brazil. One problem when screening hydrolytic activities is that the activity against commercial substrates, such as carboxymethylcellulose, does not always correspond to the activity against the natural lignocellulosic material. Besides that, the macroscopic characteristics of the raw material, such as insolubility and heterogeneity, hinder its use for high throughput screenings. RESULTS: In this paper, we present the preparation of a colloidal suspension of particles obtained from sugarcane bagasse, with minimal chemical change in the lignocellulosic material, and demonstrate its use for high throughput assays of hydrolases using Brazilian termites as the screened organisms. CONCLUSIONS: Important differences between the use of the natural substrate and commercial cellulase substrates, such as carboxymethylcellulose or crystalline cellulose, were observed. This suggests that wood feeding termites, in contrast to litter feeding termites, might not be the best source for enzymes that degrade sugarcane biomass.
RESUMO
Spodoptera frugiperda ß-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against ß-1,3-glucan (laminarin), but cannot hydrolyze yeast ß-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive ß-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of ß-1,3-glucanases and ß-glucan-binding protein supports the assumption that the ß-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived ß-1,3-glucanases by the loss of an extended N-terminal region and the ß-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.
Assuntos
Proteínas de Transporte/metabolismo , Glucana 1,3-beta-Glucosidase/química , Glucana 1,3-beta-Glucosidase/metabolismo , Proteínas de Insetos/metabolismo , Lectinas/metabolismo , Spodoptera/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Trato Gastrointestinal/química , Trato Gastrointestinal/metabolismo , Glucana 1,3-beta-Glucosidase/isolamento & purificação , Glucanos , Proteínas de Insetos/química , Larva/enzimologia , Lectinas/química , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Alinhamento de Sequência , Spodoptera/crescimento & desenvolvimentoRESUMO
Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease, a devastating disease that disables and leads to the death of many people in Latin America. In this review, factors from the insect vector are described, including digestive enzymes, hemolysins, agglutinins, microbiota and especially antimicrobial factors, which are potentially involved in regulating the development of T. cruzi in the gut. Differential regulation of parasite populations shows that some triatomine defense reactions discriminate not only between molecular signals specific for trypanosome infections but also between different strains of T. cruzi.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Triatominae/fisiologia , Triatominae/parasitologia , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/patogenicidade , Animais , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Vetores de Doenças , Humanos , Estágios do Ciclo de Vida , Triatominae/enzimologiaRESUMO
The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.
Assuntos
Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Tenebrio/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Celulases/química , Celulases/metabolismo , Clonagem Molecular , Sequência Consenso , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Trato Gastrointestinal/enzimologia , Regulação Enzimológica da Expressão Gênica , Glucana Endo-1,3-beta-D-Glucosidase/química , Concentração de Íons de Hidrogênio , Larva/enzimologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Alinhamento de Sequência , Tenebrio/classificação , Tenebrio/genéticaRESUMO
Insects are exposed to a wide range of microorganisms (bacteria, fungi, parasites and viruses) and have interconnected powerful immune reactions. Although insects lack an acquired immune system they have well-developed innate immune defences that allow a general and rapid response to infectious agents.Over the last few decades we have observed a dramatic increase in the knowledge of insect innate immunity, which relies on both humoral and cellular responses. However, innate reactions to natural insect pathogens and insect-transmitted pathogens, such as parasites, still remain poorly understood.In this review, we briefly introduce the general immune system of insects and highlight our current knowledge of these reactions focusing on the interactions of Trypanosoma rangeli with Rhodnius prolixus, an important model for innate immunity investigation.
RESUMO
In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 degrees C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 degrees C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.