RESUMO
The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and ß-fructofuranosidases (invertases) were identified. Genes encoding ß-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.
Assuntos
Dípteros , Larva , Glândulas Salivares , Animais , Glândulas Salivares/metabolismo , Glândulas Salivares/enzimologia , Larva/genética , Larva/metabolismo , Larva/crescimento & desenvolvimento , Dípteros/genética , Dípteros/enzimologia , Dípteros/metabolismo , Transcriptoma , Digestão , Genômica , Trato Gastrointestinal/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genéticaRESUMO
OBJECTIVE: Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. RESULTS: First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings.
Assuntos
Dípteros , Larva , Serina Proteases , Animais , Dípteros/genética , Dípteros/metabolismo , Dípteros/enzimologia , Larva/metabolismo , Larva/genética , Cavalos , Serina Proteases/metabolismo , Serina Proteases/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , FezesRESUMO
Background: Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity has a major issue for long-term treatment. One method to improve therapies and methods is the application of immunomodulatory agents from parasites such as Hypoderma lineatum. Hypodermin A (HA) is a serine esterase secreted by the larvae of Hypoderma lineatum, several studies demonstrated its immunosuppressive mechanism in vitro, and recently we discovered that HA inhibits the expression of interferon (IFN)-γ and interleukin (IL)-2 and activates IL-10 expression. Therefore, we hypothesized that it might be a potential agent used to block allograft rejections. However, most studies of the immunosuppressive mechanisms associated with HA were undertaken at the cellular level. In order to augment these studies, we evaluated the immunosuppressive effects of HA in vivo using an HA transgenic mouse model. Result: Our results revealed similar findings to those reported by in vitro studies, specifically that HA induced prostaglandin E2 expression, downregulated IFN-γ and IL-2 expression, and promoted IL-10 secretion via E-type prostanoid receptor 4. Additionally, we observed that HA overexpression inhibited lipopolysaccharide-induced TLR4 activation. These findings provide insight into a new potential agent capable of blocking graft rejection. Conclusion: Our founding suggested that HA-related treatment could be a promising option to improve the viability of grafts in human.
Assuntos
Animais , Camundongos , Serina Endopeptidases/imunologia , Dípteros/enzimologia , Dípteros/imunologia , Rejeição de Enxerto/imunologia , Ensaio de Imunoadsorção Enzimática , Serina Endopeptidases/metabolismo , Western Blotting , Citocinas , Terapia de Imunossupressão , Interleucinas/antagonistas & inibidores , Interferons/antagonistas & inibidores , Interleucina-10/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like , Reação em Cadeia da Polimerase em Tempo Real , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/prevenção & controleRESUMO
Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3' end, and 5' end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3' end, and 5' end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions: MMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.
Assuntos
Glândulas Salivares/enzimologia , Metaloproteinase 1 da Matriz/genética , Dípteros/enzimologia , Dípteros/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , DNA Complementar/genética , Biologia Computacional , LarvaRESUMO
Introducción. Las úlceras crónicas son una afección con un impacto negativo importante en la calidad de vida de los pacientes y en el sistema de salud; la aparición de infecciones y su difícil manejo, así como la presencia de tejido necrótico, afectan el pronóstico de curación. La larvaterapia se presenta como una opción para el desbridamiento y el manejo de infecciones de úlceras crónicas. Objetivo. Evaluar la larvaterapia en heridas con poca carga de tejido necrótico y evaluar las excreciones, secreciones y la hemolinfa de las larvas, respecto a su contenido enzimático. Materiales y métodos. Se reporta una serie de tres casos clínicos con úlceras crónicas y poca carga de tejido necrótico, tratados con larvaterapia, y se evalúa su evolución por los índices PUSH (Pressure Ulcer Scale for Healing) y Wound Bed Score, así como el patrón electroforético y contenido enzimático por zimograma de las excreciones y secreciones, y de la hemolinfa de las larvas. Resultados. Con solo una aplicación de la larvaterapia se evidenció una mejoría del aspecto de la herida y en los puntajes evaluados; en el PUSH hubo una disminución de 2,3 puntos, en promedio, y con el Wound Bed Score, un incremento de 2,7, lo que demuestra una mejoría en ambas escalas. Conclusión. Se encontró una actividad enzimática diversa en su contenido de excreciones y secreciones, con predominio de actividad de la proteasa de tipo serina.
Introduction. Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. Objective. The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. Results. The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demontrated with major activity belonging to serine protease family. Conclusions. Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.
Assuntos
Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Terapia Biológica , Desbridamento/métodos , Pé Diabético/terapia , Proteínas de Insetos/análise , Larva/enzimologia , Peptídeo Hidrolases/análise , Úlcera Varicosa/terapia , Antibacterianos/uso terapêutico , Terapia Combinada , Pé Diabético/patologia , Dípteros/enzimologia , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Hemolinfa/enzimologia , Proteínas de Insetos , Necrose , Manejo da Dor , Peptídeo Hidrolases , Índice de Gravidade de Doença , Úlcera Varicosa/tratamento farmacológico , Úlcera Varicosa/patologia , CicatrizaçãoRESUMO
Knowledge of the carrion-breeding insects present at a local level is important and necessary for defining the post-mortem interval. Climate changes and globalisation are affecting species ranges and population dynamics. In this note, we report the incidence of Chrysomya albiceps (Diptera: Calliphoridae) on dead human bodies and carrion in Northern Italy. These data confirm the spread of this species in the Northern regions. The partial sequencing of a 583-bp region of the cytochrome oxidase subunit 1 gene of an Adriatic population did not reveal any difference compared to the same genomic region in the African and South American populations of this species.
Assuntos
Adulto , Animais , Feminino , Humanos , Pessoa de Meia-Idade , Dípteros/crescimento & desenvolvimento , Complexo IV da Cadeia de Transporte de Elétrons/genética , Medicina Legal , Cadáver , Dípteros/enzimologia , Itália , Larva/enzimologia , Larva/crescimento & desenvolvimento , Dinâmica Populacional , SuínosRESUMO
We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.
Assuntos
Animais , Dípteros/enzimologia , Serina Endopeptidases/metabolismo , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Larva/enzimologia , Serina Endopeptidases/isolamento & purificaçãoRESUMO
Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of naïve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.
Assuntos
Animais , Dípteros/enzimologia , Dípteros/imunologia , Dípteros/metabolismo , Óxido Nítrico Sintase/metabolismo , Saccharomyces cerevisiae/imunologia , Larva/enzimologia , Larva/imunologia , Larva/metabolismo , Distribuição TecidualRESUMO
Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses, which include both cellular and humoral components. Cellular responses are mediated by hemocytes, and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In mammals, nitric oxide synthases (NOSs) are also present in the endothelium, the brain, the adrenal glands, and the platelets. Studies on the distribution of NO-producing systems in invertebrates have revealed functional similarities between NOS in this group and vertebrates. We attempted to localize NOS activity in tissues of na´ve (UIL), yeast-injected (YIL), and saline-injected (SIL) larvae of the blowfly Chrysomya megacephala, using the NADPH diaphorase technique. Our findings revealed similar levels of NOS activity in muscle, fat body, Malpighian tubule, gut, and brain, suggesting that NO synthesis may not be involved in the immune response of these larval systems. These results were compared to many studies that recorded the involvement of NO in various physiological functions of insects.(AU)
Assuntos
Animais , Dípteros/enzimologia , Dípteros/imunologia , Dípteros/metabolismo , Óxido Nítrico Sintase/metabolismo , Saccharomyces cerevisiae/imunologia , Distribuição Tecidual , Larva/enzimologia , Larva/imunologia , Larva/metabolismoRESUMO
Este estudo foi conduzido para verificar a ocorrência de forídeos parasitóides da saúva Atta laevigata (Smith) na região de Porto Nacional, TO, e para avaliar algumas características biológicas dessas moscas. Para tanto, entre outubro/2002 e setembro/2003, em intervalos de aproximadamente 15 dias, foram realizadas 22 coletas, cada uma contendo em média 149,5 ± 82,9 operárias, totalizando 3.704 formigas. No laboratório, as formigas eram distribuídas em grupos de cinco indivíduos por placa de Petri e mantidas a 25 ± 1°C e 80 ± 5 por cento UR, sendo alimentadas diariamente, até sua morte, com solução de mel a 50 por cento. Foram coletadas 106 operárias (2,8 por cento), com ou sem carga (fragmento vegetal), parasitadas por forídeos: 72 operárias (68 por cento) estavam parasitadas por Apocephalus vicosae Disney; 28 (26,4 por cento) por Neodohrniphora erthali Brown; e seis (5,6 por cento) por Apocephalus attophilus Borgmeier. As maiores taxas de parasitismo por A. vicosae (3,9-5,4 por cento) foram registradas nos meses secos de junho a agosto, enquanto que o parasitismo por N. erthali manteve-se abaixo de 2,3 por cento ao longo do ano. As operárias maiores estavam parasitadas pela espécie de maior tamanho N. erthali ou pela espécie que produz o maior número de larvas A. attophilus. O registro das três espécies de forídeos no Cerrado do Tocantins amplia a distribuição desses parasitóides no Brasil. Ao mesmo tempo, o desenvolvimento da larva de A. vicosae no tórax do hospedeiro e a ruptura entre o pronoto e a propleura da formiga para a emergência desta mosca são características inéditas do parasitismo de saúvas por forídeos.
The purpose of this study was to verify the occurrence of parasitism in Atta laevigata (Smith) by phorids in Porto Nacional, Tocantins, Brazil and to evaluate some biological characteristics of those flies. From October/2002 to September/2003, at 15-day intervals, 22 samplings were carried out (149.5 ± 82.9 workers each) comprising 3,704 ants. In the laboratory, the ants were distributed in groups of five individuals per petri dish and maintained at temperature of 25 ± 1°C and 80 ± 5 percent of RH. They were fed daily with a 50 percent honey solution until death. The total of 106 workers (2.8 percent), collected with or without load (plant fragment), were parasitized by phorids: 72 workers (68 percent) were parasitized by Apocephalus vicosae Disney, 28 individuals (26.4 percent) by Neodohrniphora erthali Brown, and six (5.6 percent) by Apocephalus attophilus Borgmeier. The higher parasitism rates by A. vicosae (3.9-5.4 percent) were observed during the dry season (June-August), while the parasitism by N. erthali was lower than 2.3 percent throughout the year. The bigger workers were parasitized by bigger parasitoid N. erthali or by parasitoid with larger number of larvae A. attophilus. The occurrence of three phorid species in Tocantins broadens the distribution of these flies in Brazil. The development of A. vicosae larva within the thorax of the host as well as the rupture between the ant pronotum and propleuron so that the emergence of the fly can take place are characteristics not yet described on parasitism of leaf-cutting ants by phorids.
Assuntos
Himenópteros/crescimento & desenvolvimento , Himenópteros/enzimologia , Dípteros/crescimento & desenvolvimento , Dípteros/enzimologia , Dípteros/parasitologiaRESUMO
Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As Iisozimas digestivas de insetos são do tipo "c" e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pK IND.as do nucleófilo e do doador de prótons...