RESUMEN
Sphingolipids and their metabolites have been implicated in viral infection and replication in mammal cells but how their metabolizing enzymes in the host are regulated by viruses remains largely unknown. Here we report the identification of 12 sphingolipid genes and their regulation by Rice stripe virus in the small brown planthopper (Laodelphax striatellus Fallén), a serious pest of rice throughout eastern Asia. According to protein sequence similarity, we identified 12 sphingolipid enzyme genes in L. striatellus. By comparing their mRNA levels in viruliferous versus nonviruliferous L. striatellus at different life stages by qPCR, we found that RSV infection upregulated six genes (LsCGT1, LsNAGA1, LsSGPP, LsSMPD4, LsSMS, and LsSPT) in most stages of L. striatellus Especially, four genes (LsCGT1, LsSMPD2, LsNAGA1, and LsSMS) and another three genes (LsNAGA1, LsSGPP, and LsSMS) were significantly upregulated in viruliferous third-instar and fourth-instar nymphs, respectively. HPLC-MS/MS results showed that RSV infection increased the levels of various ceramides, such as Cer18:0, Cer20:0, and Cer22:0 species, in third and fourth instar L. striatellus nymphs. Together, these results demonstrate that RSV infection alters the transcript levels of various sphingolipid enzymes and the contents of sphingolipids in L. striatellus, indicating that sphingolipids may be important for RSV infection or replication in L. striatellus.
Asunto(s)
Regulación de la Expresión Génica , Hemípteros/genética , Hemípteros/virología , Proteínas de Insectos/genética , Esfingolípidos/genética , Tenuivirus/fisiología , Animales , Cromatografía Líquida de Alta Presión , Femenino , Hemípteros/enzimología , Hemípteros/metabolismo , Proteínas de Insectos/metabolismo , Masculino , Ninfa/enzimología , Ninfa/genética , Ninfa/metabolismo , Ninfa/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Esfingolípidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Most living organisms developed the innate clock system to anticipate daily environmental changes and to enhance their chances of survival. timeless (tim) is a canonical clock gene. It has been extensively studied in Drosophila melanogaster (Diptera: Drosophilidae) as a key component of the endogenous circadian clock, but its role is largely unknown in some agriculture pests. Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae), an important rice pest, exhibits a robust locomotor rhythm. In the present study, we cloned tim gene (ls-tim) from L. striatellus and investigated its function in the regulation of behavioral rhythms. Quantitative real-time polymerase chain reaction revealed a circadian expression pattern of ls-tim under different light conditions with a trough in the photophase and a peak in the late scotophase. After the knockdown of ls-tim via RNA interference (RNAi), the adults showed an earlier onset of locomotor activity under light/dark cycles and became arrhythmic in constant darkness. ls-tim RNAi also abolished the timing of adult emergence that normally occurs in the early photophase. These results suggest that ls-tim is essential for the light-entrained circadian rhythms in L. striatellus and provide more insights into the endogenous clock network underlying the behavioral and physiological rhythms of this insect.
Asunto(s)
Relojes Circadianos/genética , Ritmo Circadiano/genética , Hemípteros/genética , Proteínas de Insectos/genética , Animales , Proteínas de Drosophila/genética , Femenino , Hemípteros/metabolismo , Proteínas de Insectos/metabolismo , Locomoción , Masculino , Fotoperiodo , Interferencia de ARN , Análisis de Secuencia de ADNRESUMEN
Most living organisms have developed internal circadian clocks to anticipate the daily environmental changes. The circadian clocks are composed of several transcriptional-translational feedback loops, in which cryptochromes (CRYs) serve as critical elements. In insects, some CRYs act as photopigments to control circadian photoentrainment, while the others act as transcriptional regulators. We cloned and characterized two cryptochrome genes, the Drosophila-like (lscry1) and vertebrate-like (lscry2) genes, in a rice pest Laodelphax striatellus. Quantitative real-time PCR showed that lscry1 and lscry2 expressed ubiquitously from nymph to adult stages as well as in different tissues. The transcript levels of lscry2 fluctuated in a circadian manner. Constant light led to arrhythmic locomotor activities in L. striatellus. It also inhibited the mRNA oscillation of lscry2 and promoted the transcription of lscry1. Knockdown of lscry1 or lscry2 by RNA interference (RNAi) reduced the rhythmicity of L. striatellus in constant darkness, but not in light dark cycles. These results suggested that lscry1 and lscry2 were putative circadian clock genes of L. striatellus, involved in the regulation of locomotor rhythms.