RESUMO
Carboxylesterases (CarEs) belong to a superfamily of metabolic enzymes encoded by a number of genes and are widely distributed in microbes, plants and animals including insects. These enzymes play important roles in detoxification of insecticides and other xenobiotics, degradation of pheromones, regulation of neurodevelopment, and control of animal development. In this study, we characterized a total of 39 full-length cDNAs putatively encoding different CarEs from the migratory locust, Locusta migratoria, one of the most severe insect pests in many regions of the world, and evaluated the role of four CarE genes in insecticide detoxification. Our phylogenetic analysis grouped the 39 CarEs into five different clades including 20 CarEs in clade A, 3 in D, 13 in E, 1 in F and 2 in I. Four CarE genes (LmCesA3, LmCesA20, LmCesD1, LmCesE1), representing three different clades (A, D and E), were selected for further analyses. The transcripts of the four genes were detectable in all the developmental stages and tissues examined. LmCesA3 and LmCesE1 were mainly expressed in the fat bodies and Malpighian tubules, whereas LmCesA20 and LmCesD1 were predominately expressed in the muscles and hemolymph, respectively. The injection of double-stranded RNA (dsRNA) synthesized from each of the four CarE genes followed by the bioassay with each of four insecticides (chlorpyrifos, malathion, carbaryl and deltamethrin) increased the nymphal mortalities by 37.2 and 28.4% in response to malathion after LmCesA20 and LmCesE1 were silenced, respectively. Thus, we proposed that both LmCesA20 and LmCesE1 played an important role in detoxification of malathion in the locust. These results are expected to help researchers reveal the characteristics of diverse CarEs and assess the risk of insecticide resistance conferred by CarEs in the locust and other insect species.
Assuntos
Hidrolases de Éster Carboxílico/genética , DNA Complementar/genética , Locusta migratoria/genética , Ninfa/genética , Animais , Carbaril/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Clorpirifos/farmacologia , DNA Complementar/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Locusta migratoria/efeitos dos fármacos , Locusta migratoria/metabolismo , Malation/farmacologia , Nitrilas/farmacologia , Ninfa/efeitos dos fármacos , Ninfa/metabolismo , Piretrinas/farmacologiaRESUMO
Hemocyanins are copper-containing (Cu(+)) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Hemocianinas/genética , Proteínas de Insetos/genética , Locusta migratoria/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Teorema de Bayes , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Estágios do Ciclo de Vida/genética , Locusta migratoria/crescimento & desenvolvimento , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.