RESUMO
Pararamosis, also known as Pararama-associated phalangeal periarthritis, is a neglected tropical disease primarily affecting rubber tappers in the Amazon region. It is caused by contact with the urticating hairs of the Premolis semirufa moth caterpillar, which resides in rubber plantations. The condition is marked by the thickening of the articular synovial membrane and cartilage impairment, features associated with chronic synovitis. Given the significance of synovial inflammation in osteoarticular diseases, in this study, the role of synoviocytes and their interactions with macrophages and chondrocytes are examined when stimulated by Pararama toxins. Synoviocytes and macrophages treated with Pararama hair extract showed an increased production of cytokines IL-6, IL-1ß, and TNF-α, indicating a direct effect on these cells. In cocultures, there was a significant rise in inflammation, with levels of IL-1ß, IL-6, and chemokines CCL2, CCL5, and CXCL8 increasing up to seven times compared to monocultures. Additionally, matrix-degrading enzymes MMP-1 and MMP-3 were significantly elevated in cocultures. Chondrocytes exposed to the extract also produced IL-6, CCL2, and CCL5, and in cocultures with synoviocytes, there was a notable increase in IL-6, CCL5, and CXCL8, as well as a doubling of MMP-1 and MMP-3 levels. These findings underscore the critical role of cell crosstalk in the inflammatory and catabolic processes associated with pararamosis and demonstrate how Pararama hair extract can influence factors affecting cartilage health, providing valuable insights into this condition.
Assuntos
Condrócitos , Sinoviócitos , Animais , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Humanos , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/induzido quimicamente , Membrana Sinovial/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Doenças Negligenciadas , Técnicas de Cocultura , Células CultivadasRESUMO
Paracoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN's dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs' N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes' fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes.
Assuntos
Mariposas , Paracoccidioides , Paracoccidioidomicose , Animais , Camundongos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Virulência , Larva , Paracoccidioidomicose/microbiologia , Paracoccidioides/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mariposas/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Lepidopteran pests are major factors limiting soybean productivity in South America. In some cases, effective management of these species requires the use of foliar insecticides. For sustainable use of these insecticides, they should only be applied when insect population size exceeds an economic threshold. Since this estimation requires to determine the consumption of different species, this work aimed to integrate all these factors, studying the consumption of small (less than 1 cm long) and medium (1 to 1.5 cm long) size larvae of major lepidopteran pests to vegetative and reproductive tissues on Bt (M7739IPRO variety, containing the event MON87701 which expresses the Cry1Ac protein from Bacillus thuringiensis) and non-Bt (BMX Desafio RR variety) soybeans. The feeding injury to vegetative tissues was tested in detached-leaf assays in grow chambers, and for reproductive structures the study was conducted in greenhouse with infestations at early (flowering) and mid reproductive (mid grain filling) stages. Based on the feeding behavior of the species tested, they were cast in four groups: a) Anticarsia gemmatalis and Chrysodeixis includens, defoliating only the RR variety with the lowest consumption of foliar area; b) Spodoptera eridania, defoliating both RR and IPRO varieties, consuming twice than the species mentioned above; c) Helicoverpa armigera, defoliating and being the most damaging species to pods in the RR variety; and d) S. cosmioides and S. frugiperda, defoliating and damaging pods in both varieties. The species differed in their ability to feed on IPRO varieties, so a different economic threshold should be considered. Consequently, in cases where more than one species are found simultaneously, the species composition should be considered in estimating the economic threshold. Additionally, our findings may contribute to a better decision-making to control insect feeding injury in IPRO varieties, because a slower larval growth provides more time to ensure the need of control with insecticides. In summary, this clasification contributes to an improved recommendation of sustainable insecticide use, taking into account the behavior of each species that are major soybeans pests in South America.
Assuntos
Inseticidas , Mariposas , Animais , Glycine max/genética , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/genética , Endotoxinas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Bactérias/genética , Mariposas/metabolismo , Larva , América do Sul , Controle Biológico de VetoresRESUMO
The caterpillar of the Premolis semirufa moth, commonly called Pararama, is found in the Brazilian Amazon region. Contact with the hairs can cause a chronic inflammatory reaction, termed "pararamosis". To date, there is still no specific treatment for pararamosis. In this study, we used a whole human blood model to evaluate the involvement of the complement in the proinflammatory effects of P. semirufa hair extract, as well as the anti-inflammatory potential of complement inhibitors in this process. After treatment of blood samples with the P. semirufa hair extract, there was a significant increase in the generation of soluble terminal complement complex (sTCC) and anaphylatoxins (C3a, C4a, and C5a), as well as the production of the cytokines TNF-α and IL-17 and the chemokines IL-8, RANTES, MIG, MCP-1, and IP-10. The inhibition of C3 with compstatin significantly decreased IL-17, IL-8, RANTES, and MCP-1 production. However, the use of the C5aR1 antagonist PMX205 promoted a reduction in the production of IL-8 and RANTES. Moreover, compstatin decreased CD11b, C5aR1, and TLR2 expression induced by P. semirufa hair extract in granulocytes and CD11b, TLR4, and TLR2 in monocytes. When we incubated vascular endothelial cells with extract-treated human plasma, there was an increase in IL-8 and MCP-1 production, and compstatin was able to decrease the production of these chemokines. C5aR1 antagonism also decreased the production of MCP-1 in endothelial cells. Thus, these results indicate that the extract of the Pararama bristles activates the complement system and that this action contributes to the production of cytokines and chemokines, modulation of the expression of surface markers in leukocytes, and activation of endothelial cells.
Assuntos
Mariposas , Animais , Humanos , Mariposas/metabolismo , Interleucina-17/efeitos adversos , Peçonhas , Interleucina-8 , Células Endoteliais/metabolismo , Floresta Úmida , Receptor 2 Toll-Like , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/induzido quimicamente , Proteínas do Sistema Complemento , QuimiocinasRESUMO
Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.
Assuntos
Glycine max , Mariposas , Animais , Glycine max/genética , Glycine max/metabolismo , Inibidores de Proteases/farmacologia , Regulação para Cima , Serina Proteases/metabolismo , Mariposas/genética , Mariposas/metabolismo , Quimotripsina/genética , Quimotripsina/metabolismo , Tripsina/metabolismo , Larva/genética , Larva/metabolismo , Serina/metabolismoRESUMO
The design and production of molecules capable of mimicking the binding or/and functional sites of proteins inhibitors represent a promising strategy for the exploration and modulation of gut trypsin function in insect pests, specifically Lepidoptera. Here, for the first time, we characterized the trypsin activity present in the gut, performance and development of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae when exposed to arginine-containing dipeptides. In silico assessment showed that arginine-containing dipeptides have a greater affinity for the active site of A. gemmatalis trypsins than lysine-containing peptides due to the presence of the double-charged guanidinium group that enhances the interaction at the S1 subsite of trypsins. Furthermore, the inhibitory and anti-insect potential of the peptides was demonstrated through kinetic and larval life cycle parameters, respectively. These dipeptides showed structural stability, binding to the active site, corroborated in vitro (competitive inhibition), and significant reduction of trypsin enzyme activity in the gut, survival, and weight of the A. gemmatalis larvae. Our findings reinforce the idea that small peptides are promising candidates for lepidopteran pest management. The optimization of DI2 and DI1 peptides, enhancing uptake and affinity to trypsins, may turn the use of these molecules feasible in agriculture.
Assuntos
Fabaceae , Mariposas , Animais , Arginina/farmacologia , Dipeptídeos/farmacologia , Insetos , Larva/metabolismo , Mariposas/metabolismo , Glycine max/metabolismo , TripsinaRESUMO
The saturniid genus Hylesia is well known for the cutaneous lepidopterism induced by airborne setae on contact with the skin. Although several cases of such dermatitis have been reported in Argentina, no information about their venoms and toxicological implications on human health is available yet. Thus, we conducted a morphological analysis of the setae/spines and a toxinological characterization (through biological assays and proteomic techniques) of the bristle extract from caterpillars and moths of Hylesia sp. from Misiones, Argentina. By scanning electron microscopy, we revealed the various and distinctive types of urticating structures: harpoon-shaped or spiny setae in caterpillars, and setae with barb-like structures in female moths. Their venom electrophoretic profiles were substantially different, presenting proteins related to toxicity, such as serpins and serine peptidases. The female moth venom exhibited higher caseinolytic activity than the caterpillar venom, and coincidentally only the former noticeably hydrolyzed fibrinogen and gelatin. In addition, the female venom displayed a dose-dependent procoagulant effect. The injection of this venom into mouse skin led to the rapid detection of an increased number of intact and degranulated mast cells in the dermis; a few areas of focal subcutaneous hemorrhage were also observed after 5 h of injection. Altogether, this study provides relevant information about the pathophysiological mechanisms whereby Hylesia sp. from northeastern Argentina can induce toxicity on human beings, and paves the way for treatment strategies of accidents caused by this saturniid lepidopteran.
Assuntos
Mariposas , Peçonhas , Animais , Argentina , Feminino , Camundongos , Mariposas/metabolismo , Proteômica , Saúde Pública , Peçonhas/metabolismoRESUMO
OBJECTIVE: We aimed to define the safety and toxicity of both isolated and embedded cinnamaldehyde using a pharmaceutical formulation for the treatment of oral fungal infections in an in vivo study. MATERIALS AND METHODS: Acute toxicity was assessed in studies with Galleria mellonella larvae and Danio rerio embryos (zebrafish), and genotoxicity was assessed in a mouse model. The pharmaceutical formulation (orabase ointment) containing cinnamaldehyde was evaluated for verification of both in vitro antifungal activity and toxicity in keratinized oral rat mucosa. RESULTS: In Galleria mellonella larvae, cinnamaldehyde was not toxic up to the highest dose tested (20 mg/kg) and presented no genotoxicity up to the dose of 4 mg/kg in the model using mice. However, it was found to be toxic in zebrafish embryos up to a concentration of 0.035 µg/mL; LC50 0.311; EC50 0.097 (egg hatching delay); and 0.105 (Pericardial edema). In the orabase antifungal susceptibility test, cinnamaldehyde exhibited activity in concentrations greater than 200 µg/mL. As for safety in the animal model with rats, the orabase ointment proved to be safe for use on keratinized mucosa up to the maximum concentration tested (700 µg/mL). CONCLUSIONS: At the concentrations tested, cinnamaldehyde was not toxic in vertebrate and invertebrate animal models and did not exhibit genotoxic activity. In addition, when used in the form of an ointment in orabase, having already recognized antifungal activity, it was shown to be safe up to the highest concentration tested.
Assuntos
Acroleína/análogos & derivados , Micoses/tratamento farmacológico , Acroleína/metabolismo , Acroleína/farmacologia , Acroleína/toxicidade , Animais , Antifúngicos/farmacologia , Carboximetilcelulose Sódica/análogos & derivados , Carboximetilcelulose Sódica/farmacologia , Larva/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos/embriologia , Mariposas/metabolismo , Ratos , Ratos Wistar/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
The tomato leaf miner, Tuta absoluta (Meyrick), is the most important pest for tomato production in Iran. The effect of flubendiamide and thiocyclam hydrogen oxalate insecticides was assessed on cellular energy allocation (CEA) of the third instar larvae of T. absoluta ingesting six different tomato cultivars. Plant leaves were treated with LC50 concentration of both insecticides and their energy available (Ea), energy consumption (Ec), and CEA were calculated. The results showed that total energy reserves (protein, carbohydrate, and lipid budgets) were significantly reduced after exposure to insecticides and tomato secondary metabolites. The larvae fed on Riogrande and Super Chief cultivars showed the least amount of energy available after treatment with both insecticides. The highest rate of oxygen consumption was observed in larvae fed on Calj, Super Luna, and Super strain B after treatment with flubendiamide. The amount of CEA decreased in treated larvae compared to untreated larvae. This reduction was statistically higher in treated larvae that fed on Riogrande and Super strain B cultivars. Reduction in CEA is probably due to the expenses of dealing with detoxification of insecticides and plant metabolites. CEA is a suitable and primary biomarker for the effects of cultivars and insecticides as integrate and summarizes insect energy allocation in variable situations.
Assuntos
Benzamidas , Compostos Heterocíclicos com 1 Anel , Inseticidas , Mariposas , Solanum lycopersicum , Sulfonas , Animais , Larva , Mariposas/metabolismo , OxalatosRESUMO
Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.
Assuntos
Cartilagem/patologia , Condrócitos/fisiologia , Inflamação/imunologia , Artropatias/imunologia , Osteoartrite/genética , Animais , Venenos de Artrópodes/metabolismo , Células Cultivadas , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Artropatias/induzido quimicamente , Mariposas/metabolismo , Floresta Úmida , Transdução de SinaisRESUMO
Insecticidal toxins from Bacillus thuringiensis (Bt) are valuable tools for pest management worldwide, contributing to the management of human disease insect vectors and phytophagous insect pests of agriculture and forestry. Here, we report the effects of dual and triple Bt toxins expressed in transgenic cotton cultivars on the fitness and demographic performance of Helicoverpa zea (Boddie)-a noctuid pest, known as cotton bollworm and corn earworm. Life-history traits were determined for individuals of three field populations from a region where H. zea overwintering is likely. Triple-gene Bt cotton cultivars that express Cry and Vip3Aa toxins killed 100% of the larvae in all populations tested. In contrast, dual-gene Bt cotton that express Cry1Ac+Cry1F and Cry1Ac+Cry2Ab allowed population growth with the intrinsic rate of population growth (rm) 38% lower than on non-Bt cotton. The insects feeding on Bt cotton plants that express Cry1Ac+Cry2Ab, Cry1Ac+Cry1F, or Cry1Ab+Cry2Ae exhibited reduced larval weight, survival rate, and increased development time. Additionally, fitness parameters varied significantly among the insect populations, even on non-Bt cotton plants, likely because of their different genetic background and/or previous Bt toxin exposure. This is the first report of the comparative fitness of H. zea field populations on dual-gene Bt cotton after the recent reports of field resistance to certain Bt toxins. These results document the population growth rates of H. zea from an agricultural landscape with 100% Bt cotton cultivars. Our results will contribute to the development and validation of resistance management recommendations.
Assuntos
Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Gossypium/parasitologia , Proteínas Hemolisinas/metabolismo , Mariposas/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/parasitologia , Animais , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Feminino , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Gossypium/genética , Gossypium/metabolismo , Proteínas Hemolisinas/genética , Masculino , Mariposas/embriologia , Mariposas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Dinâmica Populacional , Fatores de TempoRESUMO
The European grapevine moth, Lobesia botrana (Denis & Schiffermüller), is a serious pest in vineyards in North and South America. Mating disruption techniques have been used to control and monitor L. botrana on the basis of its sexual communication. This needs a well-tuned olfactory system, in which it is believed that pheromone-binding proteins (PBPs) are key players that transport pheromones in the antennae of moths. In this study, the selectivity of a PBP, named as LbotPBP1, was tested by fluorescence binding assays against 11 sex pheromone components and 6 host plant volatiles. In addition, its binding mechanism was predicted on the basis of structural analyses by molecular docking and complex and steered molecular dynamics (SMD). Our results indicate that LbotPBP1 binds selectively to sex pheromone components over certain host plant volatiles, according to both in vitro and in silico tests. Thus, chain length (14 carbon atoms) and functional groups (i.e., alcohol and ester) appear to be key features for stable binding. Likewise, residues such as Phe12, Phe36, and Phe118 could participate in unspecific binding processes, whilst Ser9, Ser56, and Trp114 could participate in the specific recognition and stabilization of sex pheromones instead of host plant volatiles. Moreover, our SMD approach supported 11-dodecenyl acetate as the best ligand for LbotPBP1. Overall, the dynamics simulations, contact frequency analysis and SMD shed light on the binding mechanism of LbotPBP1 and could overcome the imprecision of molecular docking, supporting the in vitro binding assays. Finally, the role of LbotPBP1 in the chemical ecology of L. botrana is discussed.
Assuntos
Proteínas de Transporte/genética , Proteínas de Insetos/genética , Mariposas/genética , Atrativos Sexuais/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Insetos/metabolismo , Masculino , Simulação de Acoplamento Molecular , Mariposas/metabolismo , Ligação ProteicaRESUMO
The main pheromone compound of Chilecomadia valdiviana (Lepidoptera: Cossidae) has been recently identified as (7Z,10Z)-7,10-hexadecadienal. The biosynthesis of this pheromone compound showing attributes of both Type I and Type II lepidopteran pheromones was studied by the topical application of isotope-labeled fatty acids to the pheromone gland and subsequent analysis of the gland contents (pheromone compounds and fatty acyl compounds) by gas chromatography-mass spectrometry. The deuterium label of D11-linoleic acid was incorporated into the pheromone compound and its putative acyl precursor (7Z,10Z)-7,10-hexadecadienoate, demonstrating that the pheromone compound is biosynthesized from linoleic acid by chain-shortening and further functional group transformation. Furthermore, the deuterium label of D3-stearic acid was also incorporated into the pheromone compound, which indicates that the pheromone can be synthesized de novo by C. valdiviana, as is the case for Type I lepidopteran pheromone compounds.
Assuntos
Alcadienos/metabolismo , Ácido Linoleico/metabolismo , Mariposas/metabolismo , Atrativos Sexuais/biossíntese , Ácidos Esteáricos/metabolismo , Animais , Feminino , Glândulas Odoríferas/metabolismoRESUMO
Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones. Two-dimensional (2D) gel electrophoresis showed differences in the protein patterns of infected and non-infected larvae. Hydrophobic interaction matrices adsorbed the bulk of larval proteins, thus suggesting that such matrices are inappropriate for this system. Only 0.03% and 2.9% of the total soluble protein from the infected larval extract was adsorbed to CM-Sepharose and SP-Sepharose matrices, respectively. Immobilized metal ion affinity chromatography represented a solid alternative because it bound only 1.4% of the total protein, but would increase the cost of the purification process. We concluded that cation-exchange chromatography is the best choice for easy purification of high-isoelectric-point proteins and proteins with arginine tags, since very few contaminating proteins co-eluted with our target protein.
Assuntos
Histidina , Mariposas , Nucleopoliedrovírus , Proteínas Recombinantes de Fusão , Animais , Cromatografia Líquida , Histidina/biossíntese , Histidina/química , Histidina/isolamento & purificação , Histidina/farmacologia , Larva/química , Larva/genética , Larva/metabolismo , Larva/virologia , Mariposas/química , Mariposas/genética , Mariposas/metabolismo , Mariposas/virologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Multitoxin Bt-crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory-selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126-bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip-Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine-type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa-binding ribosomal protein S2 (RpS2) were underexpressed. qRT-PCR of a subset of UniTags validated the HT-SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism.
Assuntos
Proteínas de Bactérias , Mariposas/metabolismo , Transcriptoma , Animais , Biblioteca Gênica , Resistência a Inseticidas/genética , Larva/metabolismo , Mariposas/genética , Proteínas Ribossômicas/metabolismo , Serina Proteases/metabolismoRESUMO
Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.
Assuntos
Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Receptores de Superfície Celular/metabolismo , Fosfatase Alcalina/isolamento & purificação , Fosfatase Alcalina/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Cromatografia Líquida , Sistema Digestório/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/isolamento & purificação , Larva/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/patogenicidade , Controle Biológico de Vetores , Receptores de Superfície Celular/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.
Assuntos
Aedes/efeitos dos fármacos , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insetos/metabolismo , Inseticidas , Mariposas/metabolismo , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Bioensaio/métodos , Endotoxinas/isolamento & purificação , Endotoxinas/toxicidade , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/isolamento & purificação , Larva/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Especificidade por Substrato/genética , Testes de Toxicidade/métodosRESUMO
Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we evaluated whether P. interpunctella lacking gut bacteria had differences in immune responses and susceptibility to the Bt formulation, Bactospeine. In order to clear gut bacteria, third instar larvae were reared on artificial diet containing antibiotics, or were obtained from sterilized eggs and reared under sterile conditions, and larvae were fed diets with or without Bt. Mortality was significantly lower (p<0.05) in bacteria-free larvae treated with Bt, compared with Bt-treated larvae with unaffected gut bacteria. The number of hemocytes was lower in control and Bt-treated larvae, but was significantly higher (p<0.001) in larvae treated with antibiotics and Bt, and larvae from presterilized eggs and reared on sterile diet had the highest number of hemocytes. Phenoloxidase activity was significantly lower (p<0.05) in Bt-treated larvae from presterilized eggs reared on antibiotics for 24h or in larvae reared on antibiotic-treated diets prior to Bt introduction compared with those fed control diet. Hemolin gene expression was reduced in larvae fed Bt diets compared with control and was not detected in larvae treated with antibiotics. Larvae from sterilized eggs and fed sterile diet never reached the pupal stage. Therefore, the loss of gut bacteria in P. interpunctella larvae affected the host immune response and expression of the hemolin gene, and significantly reduced susceptibility to Bt.
Assuntos
Bacillus thuringiensis/fisiologia , Microbioma Gastrointestinal , Imunidade Inata , Imunoglobulinas/genética , Proteínas de Insetos/genética , Mariposas/imunologia , Mariposas/microbiologia , Animais , Imunoglobulinas/metabolismo , Proteínas de Insetos/metabolismo , Mariposas/genética , Mariposas/metabolismoRESUMO
Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SIGNIFICANCE: The present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.
Assuntos
Dermatite/metabolismo , Proteínas de Insetos/análise , Mariposas/metabolismo , Comportamento de Nidação , Sensilas/metabolismo , Comportamento Sexual Animal , Zigoto/metabolismo , Animais , Feminino , Humanos , Inflamação/metabolismo , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Mariposas/fisiologia , Reprodução/fisiologia , Sensilas/químicaRESUMO
Chilecomadia valdiviana (Philippi) (Lepidoptera: Cossidae) is an insect native to Chile. The larval stages feed on the wood of economically important fruit tree species such as apple, pear, olive, cherry, and avocado, and also on eucalyptus. This causes weakening and, in case of severe infestation, death of the tree. We report identification of the sex pheromone produced by females of this species. Hexane extracts of the abdominal glands of virgin females were analyzed by gas chromatography (GC) with electroantennographic detection, GC coupled with mass spectrometry, and GC coupled to infrared spectroscopy. The major pheromone component was identified as (7Z,10Z)-7,10-hexadecadienal (Z7,Z10-16:Ald), and minor components present in the extracts were (Z)-7-hexadecenal and (Z)-9-hexadecenal, hexadecanal, and (9Z,12Z)-9,12-octadecadienal. Structural assignments were carried out by comparison of analytical data of the natural products and their dimethyl disulfide adducts with those of authentic reference samples. In field tests, traps baited with Z7,Z10-16:Ald captured significantly more males than control traps.