Molecular And Functional Characterization Of Antennal Esterases In Spodoptera Exigua And Spodoptera Litura And Odorant Binding Proteins In Nilaparvatalugens

In insects, the chemosensory systems have evolved to be highly sensitive during the long process of natural selection, enabling detection and discrimination among a diverse array of volatile chemicals that stimulate insect important behavioral and physiological responses such as finding of food, mate and oviposition sites, avoidance of predation, and regulation of population density. For a long time, pest control mainly depends on chemical control, which leads to not only the pest resistance to the insecticides, but also a potential threat to human health and environments. Therefore, the alternatives of pest control techniques are urgently needed. The chemosensation-based control techniques are important, and show prosperous perspective in integrated pest management. The deep understanding of the chemosensory mechanisms will undoubtedly be significant for the development of these chemosensation-based control techniques. The beet armyworm, Spodoptera exigua Hubner, the common cutworm Spodoptera. litura (Fabricius)(Lepidoptera:Noctuidae), and the brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae) are all severe pests of agriculture. Here, we reported the molecular and functional characterization of chemosensory related genes from S. exigua, S. litura and N. lugens. The main results are as follows:1. Molecular cloning, sequence analysis and expression pattern of antennal esterases from S. exiguaBy homologous cloning and transcriptomic analysis, eight antennal esterase (CXE) cDNA fragments were obtained and their full-length sequences were further achieved by Rapid Amplification of cDNA Ends (RACE) procedure from S1. exigua. The names and Genbank numbers of these CXEs were SexiCXES (HQ116561), SexiCXE10(JF728805), SexiCXEll (JF728804), SexiCXE13(HQ116560), SexiCXE14(JF728803), SexiCXE17(HQ116559), SexiCXE18(JF728802) and SexiCXE20(HQ116562) respectively. The genomic DNA sequences of the eight SexiCXE genes were also obtained, showing10introns in SexiCXE5, and two introns in the other CXEs. To gain the functional clues, the temporal expression patterns of SexiCXEs were investigated using quantitative-polymerase chain reaction (qPCR) experiments. SexiCXE10and SexiCXE17mainly expressed in antennae and little in other tissues; SexiCXE14displayed not only high expression level in antennae but also considerable expression level in proboscises. SexiCXE20mainly expressed in antennae and also highly in wings, legs, proboscises and heads; SexiCXE5and SexiCXE18displayed dominant expression in wings; SexiCXEll and SexiCXE13had no obvious differences in expression among tested tissues. The temporal pattern showed that eight SexiCXEs had higher expression in male antennae than in larval heads of the last instar. SexiCXE5, SexiCXE10and SexiCXE14showed an increasing trend from the first day to the third day of the adult, and then dropped to a lower level until the sixth day; SexiCXE117had a peak expression at two days before the eclosion. SexiCXE11and SexiCXE20displayed the peak expression at the fifth day and no fluctuation in other days. SexiCXE13showed no difference among the tested daypoints.2. Molecular cloning, sequence analysis and expression pattern of antennal esterases from S. lituraBased the homologue cloning, three SlituCXE fragments were obtained from male antennae of S. litura. Further RACE procedure was employed to obtain full-length sequences of CXE from S. litura. Their names and Genbank numbers were SlituCXE13(HQ116556), SlituCXE17(HQ116558) and SlituCXE18(HQ116557) respectively. The three SlituCXE genes were further cloned and sequenced from the genomic DNA preparation. SlituCXE17contained three introns, SlituCXE13and SlituCXE18contained two introns and all presented a similar intron/exon structure. Phylogenetic analysis showed SexiCXE14, SexiCXE17, SlituCXE17, SexiCXE18, SlituCXE18and SexiCXE20were classified into clade A (mitochondrial, cytosolic and secreted esterases); SexiCXE10and SexiCXE11were classified into clade B (dipteran a-esterases); SexiCXES into clade D (integumental/antennal esterases); SexiCXE13and SlituCXE13into clade E (β-esterases and pheromone esterases). Tissue expression pattern results showed that SlituCXE17mainly expressed in antennae, while SlituCXE17displayed dominant expression in wings; SlituCXE13had no obviously differences among tested tissues. In male antennae SlituCXE13showed the highest expression among the three SlituCXEs. For the temporal expression, three SlituCXEs had higher expression level in adult antennae than in larval heads of last instar. SlituCXE13displayed no obvious fluctuation during adult stage. SlituCXE18showed an expression peak at the third day, while. SlituCXE17had a peak expression at the first day of the adult.3. In vitro expression and purification of antennal esterases from S. exigua and S. litura11CXEs from S. exigua and S. litura were respectively constructed into insect baculovirus expression vector pFastBacl fused with N-terminal His-tag sequence in frame and successfully expressed in High-Five cells. The cell lysates for11recombinant proteins all showed an obvious band of about60kDa, and meanwhile SexiCXE10and SexiCXE14also expressed in cell supernatants, indicating that these two were secreted esterases. α/β-Naphthyl acetate assay revealed that all11CXEs had biological activity, but only five CXEs (SexiCXE10,11,13,14and SlituCXE13) could degrade at least one tested ester compound. These five CXEs were purified with a nickel affinity column. All purified proteins showed an obvious band at about60kDa and the purities were greater than95%checked by SDS-PAGE analysis.4. Measurements of kinetic parameters of antennal esterases from S. exigua and S. lituraIn order to investigate the function of these CXEs from S. exigua and S. litura for sex pheromone and other ester odors perception, first, the degradation activities of crude extracts of different tissues form S. exigua and S. litura were measured. Extracts of male antennae displayed higher activities for own sex pheromone compounds than other tested tissues in both moths. Crude extracts of male antennae from S. exigua could degrade all tested esters, and the degradation activity was positively correlated with carbon number of the esters (except for geranyl acetate). For purified enzymes, SexiCXE14displayed the most similar substrate activities with crude extracts of S. exigua than other purified enzymes, while SexiCXE10could degrade only ester compounds with carbon number less than or equal to12(except for geranyl acetate). SexiCXE13and SlituCXE13had highest degradation activities for sex pheromones among all tested enzymes. These two enzymes had high degradation activities with esters containing seven and eight carbon atoms (the percentages of degradation were above85%) and had no activity with phenyl benzoate and (Z)-3-hexenyl caproate; SexiCXEll showed low activity for most ester compounds with degradation percentage lower than50%. The measurements of kinetic parameters showed that SexiCXE13, SexiCXE14and SlituCXE13had higher affinity with own sex pheromones than other ester compounds, and SexiCXE14prefer to degrade the major compound Z9E12-14:Ac than the minor compound Z9-14:Ac when the two compound were mixed; SexiCXE10had no degradation activity with sex pheromones but had highest affinity with geranyl acetate. The optimal pH condition for enzyme activities of SexiCXE10, SexiCXE13and SexiCXE14were also measured. SexiCXE13and SexiCXE14showed highest activities at pH6.5, while SexiCXE10displayed a slow increasing trend in activity from pH6.5to pH9.0. Our results suggested that SexiCXE10functions as an enzyme for degrading the general odorants, SexiCXE14not only for sex pheromone components but also for general odorants, and other three enzymes (SexiCXEll, SexiCXE13and SlituCXE13) functions as integumental enzymes to clear out the odorants absorbed onto the insect body surface which could reducing background noise to enhance olfactory sensitivity.5. Prediction and validation of odorant binding proteins from N. lugens and other insects using Express Sequence TagsOdorant binding protein (OBP) plays important role in insect olfaction, but it is not reported in N. lugens which was an important agricultural pest. The increasing data of Express Sequence Tag (EST) supplied a good resource for finding new OBPs. To find new OBP genes, a computational pipeline was developed to use ESTs deposited in public database NCBI. In total,752,841insect ESTs were examined from54species covering eight Orders of Insecta. From these ESTs,142OBPs were identified, of which117OBPs are new including3OBPs from N. lugens (NlugOBPs). Of the117OBPs,88OBPs had complete ORFs.26OBPs from eight species of insects were randomly chosen for RT-PCR validation. As a result,22OBPs were confirmed, showing the efficiency and reliability of our prediction method.6. Sequence analysis and expression pattern of odorant binding proteins from N. lugens Based on the three NlugOBP cDNA fragments, full-length cDNAs were obtained using RACE strategy. Three NlugOBPs encoded173,143and147amino acids, respectively. Three NlugOBPs possessed typical sequence characters of "Classic OBP" and showed high identities with reported insect OBPs, although very low identities between three NlugOBPs. To find functional clues, the expression profiles were investigated by qRT-PCR, regarding to tissue, developmental stage, wing form and gender. NlugOBP1showed no much difference in expression level between nymph and adult stages. However, NlugOBP2expressed significantly higher in adult, while NlugOBP3significantly higher in nymph (except for fifth day-instar); NlugOBP2and NlugOBP3were obviously female biased in both short-and long-wing adults, whereas NlugOBP1was expressed in similar levels between females and males. All three NlugOBPs showed no much difference between long and short wing adults. Among tissues, NlugOBP2expressed high in antenna with little in other tissues; NlugOBP1dominantly expressed in antennae and also had considerable expression level in wings of both sexes; NlugOBP3expressed in high levels in antennae and abdomen. The distinct expression patterns among three NlugOBPs suggesting their functional differentiation.7. Functional research of odorant binding proteins from N. lugensTo elucidate functions of NlugOBPs, high yields of recombinant and soluble NlugOBPs were achieved using a bacterial expression system. With the purified NlugOBPs, competitive fluorescence ligand binding assays were carried out. As results, all three NlugOBPs displayed higher binding abilities for Terpenes and Ketones than other chemicals. However, NlugOBP3showed markedly higher binding ability and wider binding spectrum than the other two NlugOBPs. According to the binding abilities for rice volatiles and the expression patterns of the three NlugOBPs, it was proposed that NlugOBP3was more important than the two others in perception of rice volatile and other possible functions, thus NlugOBP3was selected for further functional study by RNAi. Compared to the insects feeding on a normal artificial diet (CK) and an exogenous dsRNA mixed diet (dsGFP), the nymphs on OBP3-dsRNA diet (dsOBP3) showed a significantly reduced mRNA expression, with about60%and80%reduction at1day and2days after the treatment, respectively. At meanwhile, dsOBP3treated nymphs showed significant decrease in ability to locate the rice plants, providing the evidence that NlugOBP3is involved in perception of rice volatiles. Surprisingly, the dsOBP3treatment also resulted in a significant increase in the nymph mortality, indicating non-functions of NlugOBP3. Therefore, NlugOBP3is a potential target gene for use in designing and developing new deterrent and lethal agent.In conclusion, we for the first time characterized11antennal esterases from S. exigua and S. litura, and three odorant binding proteins form N. lugens; investigated the expression patterns of these genes; and explored their functions in olfaction of sex pheromone compounds and host plant volatiles. The results obtained in this study provided the important bases for the elucidation of the molecular mechanisms of insect olfaction and target genes for designing and developing effective pest control agents.