| BackgroundInsects have evolved highly specialized and sensitive olfactory systems to detect numerous volatile chemicals in the environment. Olfaction plays an important role in foraging, locating hosts, finding mates, and selecting oviposition sites. The study of the molecular mechanisms underlying olfactory perception revealed that the critical event of olfactory transduction is the binding of odors to olfactory sensory neurons (OSNs) specifically mediated by odorant receptors (ORs). In general, each insect OSN expresses two ORs:one member of a family of the conventional ORs, and one member of a unique family of receptor subtypes, the olfactory co-receptor Orco (formerly called Or83b). Each insect species has only one Orco gene, which is a Drosophila Orco ortholog. Orco genes from different insects comprise the Orco family. Orco forms a heterodimeric complexe with a conventional OR to function as a chemosensor. The functions of Orco are to traffic conventional ORs to the surface of the dendritic membrane of OSNs, and maintain the conformation of heterodimeric complexes within the sensory cilia. The mutation of the Orco gene resulted in the loss of olfactory function in insect species. Therefore, Orco is a promising molecular target for developing novel pest control strategies.Lucilia sericata Meigen and Chrysomya megacephala Fabricius (Diptera:Calliphoridae) are two medically important blowfly species. Adults can mechanically transmit various pathogens to man causing human health problems. Larvae can also behave as facultative ectoparasites that cause myiasis in animals, leading to economic losses in the livestock industry. Previous studies have described many aspects of olfaction of some blowflies from extracellular levels by means of morphological, electrophysiological, behavioral and ecological techniques. However, the molecular mechanisms underlying olfactory perception in blowflies have not been fully known. ObjectiveTo study the molecular basis for odor recognition, clone OR genes and identify their functions. This provides theoretical foundation and practical guidance that will help in developing integrated pest management programs for the control of blowflies.Methods1. Two full-length Orco genes were cloned from L. sericata and C. megacephala using RT-PCR and RACE-PCR based strategies. The nucleotide sequences of these two genes were analyzed and the structural features of their deduced amino acid sequences were predicted by various bioinformatic tools.2. The expression of two blowfly Orco transcripts in different adult tissues including antennae, maxillary palps, thoraxes, abdomens, legs and ovipositors was examined by semi-quantitative RT-PCR using gene-specific primers.3. The CmegOrco mRNA distribution in the antennae and the maxillary palps of C. megacephala was mapped by in situ hybridization.4. The relative expression levels of L. sericata Orco transcript were determined by real-time PCR during different developmental stages:egg, first through third-instar larva, early pupa, and young adult.5. To test the role of blowfly Orco in odor recognition, behavioral responses of C. megacephala to food odor were examined in a Y-tube olfactometer after knockdown of its Orco by the RNAi technique.Results1. Two full-length cDNAs from L. sericata and C. megacephala were designated LserOrco and CmegOrco, respectively. The nucleotide sequences have been deposited in GenBank under the accession numbers HQ315862for LserOrco and HQ315861for CmegOrco. Both LserOrco and CmegOrco cDNAs contain a1437bp open reading frame encoding a protein of478amino acids in length. BLAST analysis revealed that these two deduced proteins not only share98%amino acid identity with each other, but also display significant sequence identities with previously identified Orco orthologs across different insect species. The alignment of two amino acid sequences with several receptor proteins from other insect species showed that the remarkable extreme conservation was found in the C-terminal region (the final164aa), where all protein sequences share nearly90%identity. Further sequence analysis showed that both proteins contain seven transmembrane domains with an intracellular N-terminus and extracellular C-terminus, in accordance with the membrane topology of other Orco orthologs.2. In both male and female tissues of L. sericata and C. megacephala, transcripts of LserOrco and CmegOrco were robustly expressed in the antennae and the maxillary palps, and weakly expressed in the female ovipositors. For non-olfactory tissues of both blowflies, a lower level of expression was observed in the legs of L. sericata, whereas a weak band was detected in the abdomens of C. megacephala.3. CmegOrco is expressed in most antennal OSNs and maxillary palp neurons. CmegOrco-positive cells are broadly distributed in the third antennal segment (flagellum) and the tip of maxillary palp, but sporadically detected in the first antennal segment (scape).4. LserOrco transcript was clearly expressed in pre-imago stages and adult tissue, including egg, larva, and female antennae, but not in early pupa (36hr after puparium formation). The expression levels of LserOrco were markedly higher in the adult female antennae than in tissues from other developmental stages, for example, a413-fold increase relative to the egg (as the calibrator). In contrast to its extremely high expression in the adult stage, LserOrco expression was at the lowest level during the egg stage, and then increased to a peak through the first two larval stages before declining in the third-instar stage.5. RT-PCR analysis for mRNA levels of CmegOrco in non-injected, water-injected, and dsRNA-injected C. megacephala at24,48, and72hr after injection showed a clear reduction of CmegOrco transcript levels in dsRNA-injected blowflies at48and72hr, as compared to water-injected and non-injected controls. Furthermore, water-injected and non-injected controls displayed almost equivalent levels of CmegOrco transcripts. This demonstrated that RNAi treatment is responsible for the observed reduction of CmegOrco mRNA levels. Behavioral assay results showed that compared with null hypothesis Ho (choices between clean air and decayed pork liver are equal), non-injected blowflies were significantly attracted to decayed pork liver (x2=16.634, df=1,p=0.000). Similarly, decayed pork liver elicited significant attraction in water-injected blowflies (x2=12.281, df=1, p=0.000). However, the chemotaxis behavioral responses of dsRNA-injected blowflies to decayed pork liver were not significantly different from a random distribution (x2=0.048, df=1, p=0.827), indicating that the knockdown of CmegOrco gene severely impaired the olfactory response of C. megacephala.Conclusion1. Two newly discovered OR genes represent the first members of the OR families of two vector insects, L. sericata and C. megacephala.2. LserOrco and CmegOrco are Drosophila Orco orthologs that have remarkably similar structural features and expression patterns as other members of the Orco family.3. RNAi technique coupled with the chemotaxis behavioral assay demonstrated that the newly obtained Orco receptor is an important molecular basis for olfactory perception in blowflies.4. RNAi technique targeting Orco genes may facilitate the development of new approaches designed to interfere with olfactory-mediated host-seeking behavior in disease-carrying blowflies. |
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