RESUMO
Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Heterópteros/genética , Proteínas de Insetos/genética , Muda , Interferência de RNA , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Heterópteros/crescimento & desenvolvimento , Heterópteros/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Masculino , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Filogenia , Alinhamento de SequênciaRESUMO
Bombyx mori nucleopolyhedrovirus (BmNPV) ORF 51 (Bm51) is a gene present in many lepidopteran NPVs, but its function is unknown. In this study, Bm51 was characterized. Transcripts of Bm51 were detected from 4.5 through 72 hour post infection (h p.i.) by RT-PCR. The corresponding protein was detected from 6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using a polyclonal antibody against Bm51. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm51 encodes a 23-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. The protein was temporarily called BV-E23. In addition immunofluorescence microscopy demonstrated that the protein was present within the cytoplasm and nuclei in virus-infected cells. In conclusion, the available data suggest that Bm51 is a functional ORF of BmNPV and encodes a protein expressed in the early stage of the infection cycle that is associated with the BV envelope.
Assuntos
Bombyx/virologia , Genes Virais , Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Núcleo Celular , Citoplasma/química , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Fatores de Tempo , Proteínas do Envelope Viral/química , Vírion/químicaRESUMO
This paper evaluates the efficacy of the bacterial larvicide bacillus sphaericus strains BS-10 and C3-41, which are isolated in China, as well as BS2362 against larvae of Culex pipiens pallens in laboratory and under field conditions. The results indicate that C3-41 has the highest toxicity with LC50 at 0.0057 ppm and the bacterial formulations are more effective in light polluted water than in heavy polluted one, but the action of B. sphaericus could persist longer in more polluted water.
