Identification And Functional Characterization Of Olfactory Relative Protein In Chrysopa Pallens

Base on a third trophic levels, predators often perceive volatiles that released by herbivore-injured plants, secretions or substances associated with prey, to locate their victims. Green lacewing, Chrysopa pallens Rambur(Neuroptera: Chrysopidae) is carnivorous during both the adult and larval stages and is an important natural enemy of many agricultural and forestry pests, such as aphids, mites, whiteflies, and the eggs and young larvae of lepidopteran insects. The larvae of C. pallens are called aphid-lions because of its large appetite. C. pallens are known as a prospective indigenous biological control agent in the Palaearctic region, including China. However, after the release in glass-houses or field crops, they tend to disperse away, possibly because they do not efficiently recognize either the habitat or the host. Study of olfactory gene function and olfactory behavior of C. pallens is imperative to enhance its efficacy in biological pest control using highly effective “push-pull” strategy, in which combinations of repellent and attractive stimuli are employed to regulate the populations of insect pests and their natural enemies. In this study, several kinds of olfactory genes putatively involved in olfactory reception in C. pallens were identified base on an antennae transcriptome analysis of C. pallens by Illumina Genome Analyzer IIx. Systematically functional studied of three key olfactory proteins were investigated, including odorant-binding proteins(OBPs), chemosensory proteins(CSPs), and sensory neuron membrane proteins(SNMPs). We have provided detailed evidences for the involvement of OBPs, CSPs, and SNMPs in C. pallens chemoperception, and the compounds that could be selected to develop slow-release agents that might attract/repel C. pallens. The main results are summarized as follows:1. Antennal morphology and sensilla of the green lacewing, C. pallenswere studied using scanning electron microscopy. The results showed that the linear antenna consists of the scape, pedicel and flagellum, which was divided into 161 single parts, individually. The whole length of the antennae and flagellum were measured about 1.5 cm, 1.44 cm, respectively. The length was proved to be much longer than most of the ones of the other insects reported till now. Four types of sensilla trichodea, three types of sensilla chaetica, and one of each type of sensilla basiconic and B?hm bristles were distinguished with the shapes and distribution of sensilla. No differences were found in the type, amount, and distribution of the sensilla between the male and female adults. The quality and distribution of each sensillum, however, were different in different segment of antenna.2. Based on an antennae transcriptome analysis by Illumina Genome Analyzer IIx, we have screened and identified a total of 74 olfactory related genes in C. pallens, including 6 odorant-binding proteins(OBPs), 10 chemosensory proteins(CSPs), 26 odorant receptors(ORs), 16 ionotropic receptors(IRs), 13 gustatory receptors(GRs), 2 sensory neuron membrane proteins(SNMPs). RT-PCR and qRT-PCR studied the different expression profiles of these genes among different tissues of C. pallens. The results indicated that most of the olfactory related genes were highly expressed in the antennae other than in the body, with some of them were female antennae-enriched expression, and others were male antennae-enriched expression.3. Full length of CDS of 2 OBP genes(CpalGOBP, CpalOBP) and 5 CSP genes(CpalCSP1, CpalCSP3, CpalCSP4, CpalCSP5, CpalCSP8) were cloned and sequenced. The calculated dissociation constant of CpalCSP1 combined with 1-NPN was 3.93 μM. In general, CpalCSP1 displayed high binding affinities(Ki < 20 μM) to majority of the 50 tested ligands, most of which were volatiles from pests, compatriots, or pest-damaged plants. Bis-(2-ethylhexyl) phthalate(Ki=5.78 μM),(-)-β-farnesene(8.74 μM), α-phellandrene(9.43 μM), and(-)-β-pinene(9.90 μM) bound strongly to CpalCSP1.4. A CpalCSP1 structural model was built based on the crystal structure of CSPMbraA6 of Mamestra brassicae, with which it shared 41% identity. The modeled CpalCSP1 3-D structure was typical of the CSPs, with six α-helices and a very short one near the carboxyl-terminus. The binding pocket was well conserved. Active site Tyr26 residue in CSPMbraA6 was confirmed to be involved in binding 12-bromo-dodecanol(BrC12OH), and corresponded to Tyr46 in CpalCSP1, which was likely involved in binding volatiles from pests, compatriots, or pest-damaged plants.5. We measured the electroantennography(EAG) responses of the male and female antennae to 50 volatiles, to confirm their biological activities. After a preliminary screening test, eight ligands including phenethyl alcohol, bis(2-ethylhexyl) phthalate, 4-ethylbenzaldehyde, valeric aldehyde, hexanal, nonanal,(-)-β-farnesene, and 3-methyl-1-butanol were selected with their depolarization above-1.0 mV at a final concentration of 1%(v/v) for a dose-dependent EAG response test. All tested compounds elicited apparent dose-dependent EAG responses in the antennae of adults of both the sexes, with higher responses at a dose of 10% except phenethyl alcohol. Among them, 6 compounds including bis(2-ethylhexyl) phthalate,(-)-β-farnesene, hexanal, nonanal, 4-ethylbenzaldehyde, and 3-methyl-1-butanol, elicited significantly higher EAG responses from female antennae than the male antennae.6. The full-length of sensory neuron membrane(SNMP) gene was obtained by RACE-PCR strategy. Bioinformatics analysis indicated that CpalSNMP2 was the double-transmembrane protein. Sequence alignment of CpalSNMP2 and SNMP2 sequences from other insects revealed that all the SNMP2 had seven conserved cysteine residues, Amino acid identity among them were 48.51 %. The phylogenetic analysis revealed that CpalSNMP1 and CpalSNMP2 were clustered into two braches named SNMP1 and SNMP2, respectively. The qRT-PCR results indicated that CpalSNMP2 was expressed in all the tissues ofC. pallens but was exclusively expressed in the antennae, furthermore, significant higher levels in the male antennae than in the female antennae were detected. Low expression levels were detected in heads and legs. The highest transcript levels were detected in 25-day-old adults antennae compared to 1-day-old adult antennae.