Molecular Analysis Of Chemosensory System And Metabolic Enzymes Of Rice Planthoppers

The chemosensory system (odorant and gustatory perception) and the metabolic enzymes play key roles in the adaptation of herbivorous insects to compounds in their ecological niches, with the former sophisticatedly regulates the insects with their behaviors like habitat searching, host finding, gathering, avoidance, and reproduction; with the later specifically regulates the compound metabolism and maintains the balance of physiological state. Parsing these two systems is now not only a hot spot in the area of insect-plant interaction, but also an important research field in exploring the new strategies in the sustainable control of agricultural pests. Recent development of large-scale sequencing technology offered a good support in the study of these two systems in the molecular level. The three rice planthoppers, Nilaparvata lugens (Stal), Laodelphax striatellus (Fallen) and Sogatella furcifera (Horvath) are the main and most destructive pests in rice-growing areas in Asia. It is a research frontier and difficult area for the understanding of the ecological adaptation of the three insect pests to their niches. With the finishing of the genomic sequencing and the transcriptome analyzing, this study carried out a systematic analysis in the two major systems of the three rice planthoppers:(1) We identified the key gene families of the chemosensory system for the Nilaparvata lugens. In this study, we identified four key gene families for this system:11odorant binding proteins (OBP),17chemosensory proteins (CSP),49odorant receptors (OR),1odorant receptor co-receptor (Orco) and10gustatory receptors (GR). N. lugens has the fewest genes in the chemosensory system compared with other insects with a known genome. As a typical monophagous insect, N. lugens has evolved a special chemosensory system to the only host rice. For instance, it has a significant reduction in the gene number expansion of the GR family, but its limited GR genes may still keep the basic taste recognition functions such as on the sweet (sugar), bitter, environmental gas and even sexual pheromone analogous; the expansion of the number in the OR and OBP family is also very simple, with the former showed a unique expansion; a significant expansion was discovered in the CSP family, and N. lugens is the first insect observed to possess more CSP genes than OBP genes, which might be useful for the understanding of the evolution for the two carrier proteins. In addition, the intron number and size of the chemosensory system genes in N. lugens are much larger than other insects.(2) We identified the key genes of the chemosensory system for Laodelphax striatellus and Sogatella furcifera from their transcriptomes, profiled the expression of these genes and analyzed the impact of the rice stripe virus on them. We identified27OBPs,31CSPs and2Orcos in the transcriptome of the three rice planthoppers. Among them, OBP and CSP are very conserved during the evolution of the three species, which might be caused by purify selection. Only a few genes we found were specifically expressed in the antennae, while most genes were aboundantly found in the heads (without antennae), legs, the remainning bodies (without heads and legs). Another support for the coevolution of the chemosensory system in the rice planthoppers is the high similarity of the Orco gene in the two species N. lugens and L. striatellus, with only a few differences in the protein level. The infection of rice stripe virus significantly improved the expression level of some OBPs and CSPs in the adult females and3rd instar nymphs of L. striatellus, and the relative responses to rice seedlings were also increased in adult female stage, but not the males.(3) We for the first time identified the glutathione S-transferase (GST) gene family of the N. lugens, and identified the GST genes in L. striatellus and S. furcifera. We profiled the expression characteristics of these genes in insect body and analyzed their alternation by ecological factors. We interferenced the GST gene expression in N. lugens and studied the impact of the rice stripe virus on them. In this study, we identified11functional genes and2pseudogenes in the GST families from N. lugens genome,9GST genes for L. striatellus and9for S. furcifera from their transcriptomes. N. lugens has the fewest genes in the GST gene family compared with other insect with a known genome, especially for the two subfamilies (Delta and Epsilon) closely related to the metabolism of xenobiotics, and the intron number and size of the GST in N. lugens are both much larger than other insects. The GST genes cover5subfamilies in three planthoppers, and some orthologous gene groups with high similarity were identified from them. Among the three species, the tissue-and life-stage-expression characteristics of these orthologous genes were much similar, but their induced expression to insecticides were different. The fold changes of the GST genes’ expression were within10folds, smaller than P450genes, and most of them were among1.5to4.5folds. RNA interference of two GST genes NIGSTel and NlGSTm2by dsRNA injection significantly improved the sensitivity of nymph to insecticides. The infection of rice stripe virus significantly improved the expression level of some GST genes (LsGSTs3) in L. striatellus, indicating that this gene family may be involved in the interaction between the insect vector and the virus.(4) We identified the cytochrome P450monooxygenase gene for the three rice planthoppers from their transcriptomes, and profiled their expression of them. In this study, we identified atotal of154P450superfamily genes in the transcriptomes of the three rice planthoppers, which was classified into4groups and16families. Among the three insects, L. striatellus and S. furcifera has the highest similarity in their orthologous genes. As to the expression level, P450genes showed a highest mRNA level in the nymphs and the macropterous adults, and lower in the eggs, and the lowest in the branchypterous adults, which might be related to the complexity and diversity of their functions. The CYP2group genes, which are closely related to the metabolism of endogenous compounds, have a constantly high expression compared to the CYP6gene family, which is related to the metabolism of xenobiotics.(5) We expressed and purified the OBP from L. striatellus, and constructed two dsRNA based transgenic rice lines with the two key genes LsGSTe and LsOrco. In this study, we constructed the expression vector of7OBP genes from L. striatellus, test the best condition for their expression, and then expressed and purified the two proteins LsOBP5and LsOBP7. With the dsRNA based transgenic rice, the L. striatellus nymphs were kept feeding in the leaf areas. After feeding for48hours and96hours on the two kinds of transgenic rice plant, the mRNA level of target genes were significantly reduced in the insects, respectively. In addition, after feeding on the transgenic rice expressing LsOrco dsRNA, the host choice behavior of L. striatellus nymphs were significantly affected and the attraction to rice seedling were significantly decreased.