RESUMO
Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions.
Assuntos
Regulação da Expressão Gênica , Estágios do Ciclo de Vida/genética , Superóxido Dismutase/genética , Tenebrio/genética , Tenebrio/parasitologia , Animais , Sequência de Bases , DNA Complementar , Infecções por Escherichia coli , Corpo Adiposo/enzimologia , Hemócitos/enzimologia , Dados de Sequência Molecular , Superóxido Dismutase/metabolismo , Tenebrio/enzimologia , Regulação para Cima , Vespas/fisiologiaRESUMO
Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.
Assuntos
Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/enzimologia , Lepidópteros/genética , Vespas/parasitologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catecol Oxidase/química , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Regulação para Baixo/efeitos dos fármacos , Precursores Enzimáticos/química , Feminino , Hemolinfa/enzimologia , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Parasita/fisiologia , Imunidade Inata , Proteínas de Insetos/química , Lepidópteros/imunologia , Lepidópteros/parasitologia , Melaninas/biossíntese , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Venenos de Vespas/toxicidadeRESUMO
Parasitism by the endoparasitoid wasp Pteromalus puparum causes alterations in the plasma proteins of Pieris rapae. Analysis of plasma proteins using a proteomic approach showed that seven proteins were differentially expressed in the host pupae after 24-h parasitism. They were masquerade-like serine proteinase homolog (MSPH), enolase (Eno), bilin-binding protein (BBP), imaginal disc growth factor (IDGF), ornithine decarboxylase (ODC), cellular retinoic acid binding protein (CRABP), and one unknown function protein. The full length cDNA sequences of MSPH, Eno, and BBP were successfully cloned using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the transcript levels of MSPH and BBP in the fat bodies of host pupae were inducible in response to the parasitism and their variations were consistent with translational changes of these genes after parasitism, while the transcript levels of Eno and IDGF were not affected by parasitism. This study will contribute to the better understanding of the molecular bases of parasitoid-induced host alterations associated with innate immune responses, detoxification, and energy metabolism.
Assuntos
Lepidópteros/parasitologia , Vespas/metabolismo , Vespas/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , Perfilação da Expressão Gênica , Genes de Insetos , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Vespas/genéticaRESUMO
Parasitoid venom is a complex mixture of active substances with diversified biological functions. Because of its range of activities, venom is an important resource with respect to potential application in agriculture and medicine. Only a limited number of peptides, proteins, and enzymes have been identified and characterized from parasitoid venom. Here we describe a proteomic analysis of the venom from the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Venom resolved by two-dimensional electrophoresis yielded 56 protein spots with major proteins in the pI range 4-7 and molecular mass range of 25-66.2 kDa. The amino acid sequences of the proteins were identified by mass spectrometry. Several venom proteins such as calreticulin, venom acid phosphatase, serine protease, arginine kinase, serine protease homolog, aminotransferase-like venom protein, and heat shock protein 70, were identified in silico based on their amino acid sequences. The full-length cDNAs of calreticulin and arginine kinase were cloned. Calreticulin showed 62% identity with calreticulin in the venom of Cotesia rubecula. Arginine kinase showed a high level of sequence identity (92%) with its counterpart in the venom of Cyphononyx dorsalis. RT-PCR analysis revealed that the transcript levels of calreticulin and arginine kinase were developmentally changed, suggesting a possible correlation with the oviposition process. This study contributes to our appreciation of a parasitoid wasp venom composition.