Cloning, Expression And Function Study Of Odorant Receptors And Sensory Neuron Membrane Proteins In Spodoptera Exigua

In insects, the relationships between individuals and the environment mainly depend on the chemical communications. With the various chemosensory organs, especially olfactory organs, insects can percept the array of environmental chemical cues and subsequently respond with various physiological and behavioral responses, such as finding habitats and partners, and escaping from prayers. Sex pheromone communication system in insects especially lepidopteran insects is extremely sensitive and species-specific, thus is thought as a perspective alternative to be exploited in pest control. However, the sex attractants currently used have many shortcomings, such as the unstable structure, and inability in attracting males, which is not ideal for those lepidopteran pests with multiple matings. These shortcomings would be overcome by the deeper understanding of molecular mechanism of insect olfactory recognition. There are many proteins involved in olfactory recognition, such as odorant binding proteins (OBPs), odorant degrading enzyme (ODEs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs). In Lepidoptera, many studies have focused on OBPs, but few on OR and SNMP. ORs located on the sensory dendritic membrane function to transform external chemical signals into electrical signals, while SNMPs may play an important role in the orientation of odorants onto and activation of the ORs. In the current study, focusing on the molecules mechanism of finding mating partners by males and habitats by both sexes in the beet armyworm, Spodoptera exigua Hubner (Lepidoptera:Noctuidae), we cloned four pheromone receptor (PR) genes (OR6, OR11, OR13and OR16), two general odorant receptor genes (OR3and OR18) and two SNMPs (SNMP1and SNMP2), and explored the their possible roles in the perception of sex pheromone or plant odorants. The main results were as follows:1. Molecular cloning, sequence analysis, expression pattern and functional characterization of pheromone receptors from S. exigua By homologous cloning, four pheromone receptors (PR) cDNA fragments were obtained and their full-length sequences were further achieved by Rapid Amplification of cDNA Ends (RACE) procedure from S. exigua. The names (GenBank numbers) of these PRs were SexiOR6(KC299916), SexiORll (KC299917), SexiOR13(KC299918) and SexiOR16(KC299919) respectively. Phylogenetic analyses reveal that all four ORs belong to pheromone receptor subtypes. The expression patterns of SexiOR6,11,13and16genes were analyzed by qPCR. The transcripts of all four PRs were expressed at high levels in antennae, of which SexiOR6,13and16are male antenna-specific, with weak, if any, expression in other olfactory tissues, such as proboscises and labial palps, while SexiORll is female antenna-biased with low expression levels in other olfactory tissues. Among the four genes in antennae. SexiOR6was expressed at approximately one order of magnitude higher than SexiORll,13and16in male antennae, which was different to those detected other insects. Functional analyses of these PRs were conducted using heterologous expression system of Xenopus oocytes. SexiOR13and SexiOR16were all broadly activated by multiple pheromone components. SexiOR13responded robustly to the ester compound Z9,E12-14:OAc and Z9-14:OAc, but SexiOR13was approximately4times sensitive to Z9,E12-14:OAc (EC5O=3.158×10-6M) compared to Z9-14:OAc (EC50=1.203×10-5M). In contrast, SexiOR16responded robustly to the alcohol component Z9-14:OH with high sensitivity (EC50=9.690×10-7M). This provides important data for further research on the molecular mechanisms of pheromone reception.2. Molecular cloning, sequence analysis, expression pattern and functional characterization of general odorant receptors from S. exiguaBy homologous cloning, two general odorant receptors (OR) cDNA fragments were obtained and their full-length sequences were further achieved by Rapid Amplification of cDNA Ends (RACE) procedure from S. exigua. The names (GenBank numbers) of these ORs were SexiOR3(JF747606) and SexiOR18(JN873314). Phylogenetic analyses revealed that the two ORs belong to general odorant receptor subtypes. qPCR measurements showed that the two ORs were expressed at high levels in antennae, with very weak, if any, expression in other olfactory tissues, such as proboscises and labial palps. Unlike PRs, their expression levels in female antenna were significant higher than male antennae. The in situ hybridization was used to explore the sensillum distribution of the two genes and Orco gene in S. exigua, and the results revealed that SexiOR3was mainly expressed under short sensilla trichoid and weakly expressed in long sensilla trichoid, while no signal was found under sensilla coeloconic and sensilla chaetica. SexiOR18was mainly expressed under sensilla trichoid and sensilla basconica,but no signal was found under sensilla coeloconic and sensilla chaetica. SexiOrco was relatively more widely expressed under sensilla trichoid, sensilla basconica and sensilla coeloconic, but no signal was found under sensilla styloconica and sensilla chaetica.Functional analyses of SexiOR3and18are performed by using heterologous expression system of Xenopus oocytes. Among62tested odorants, SexiOR3response only to E-β-farnesene, Farnesene, Farnesol, Neraniol, Geraniol and Octyl acetate. E-β-farnesene eliciated the bigest response with the EC50of3.599×10-7M. Interesting, we found that phenylacetaldehyde can significantly inhibit the response by E-β-farnesene especially at higher concentration; therefore, we suggest that phenylacetaldehyde might be an inhibitor of the recognition of E-β-farnesene by SexiORS. SexiOR18did not evoke any responses to all62general odorants, thus the function remains to be further research.3. Molecular cloning, sequence analysis and expression pattern of sensory neuron membrane proteins from S. exiguaBy homologous cloning, two sensory neuron membrane proteins (SNMPs) cDNA open reading frame were obtained from S. exigua. The names (GenBank numbers) of these SNMPs were SexiSNMP1(JX469106) and SexiSNMP2(JX469107) respectively. Phylogenetic analyses reveal that the two SNMPs belong to two separate SNMP subtypes, with a similarity of29%between each other. The expression patterns of SexiSNMP1and2genes were analyzed by qPCR. The transcripts of SexiSNMP1was antenna-specific, while SexiSNMP2was mainly expressed in antenna, with weak, if any, expression in other olfactory tissues, such as proboscises and labial palps. The expression levels of SexiSNMP2were significantly higher than SexiSNMP1in male antenna, but no significant in female. The in situ hybridization was used to explore the sensillum distributions of the two genes in both female and male antenna of S. exigua, and the results revealed that SexiSNMPl and SexiSNMP2were all expressed under sensilla trichoid and sensilla basconica, suggesting their involvment in the detection of not only the sex pheromone but also general odorants.In conclusion, we for the first time characterized6ORs and2SNMPs from the antennae of S. exigua, investigated the expression patterns of these genes, and explored their functions in olfaction of pheromone compounds and general odorants. The results obtained in this study provided the important bases for the elucidation of the molecular mechanisms of insect olfaction, and potential target genes for designing and developing effective pest control agents.