Molecular And Functional Characterization Of Chemosensory Genes In Ectropis Obliqua

In lepidopteran species, choosing the right host plant during oviposition is particularly important, because it conditions the success of their progeny. The small larvae cannot easily forage for alternate host plants. The process of host plant selection for feeding and oviposition can be regarded as a continuum which is involved in searching, landing, contact evalution and final acceptance or rejection. Semiochemicals such as host volatiles and plant secondary compounds inside plant tissues are crucial for insects to locate host plants and evaluate oviposition sites. Insects typically first locate their host plants from a distance by detection of airborne odorant molecules with their sophisticated olfactory system. Subsequent selection of the host occurs only after contact evaluation of nonvolatile chemicals in or on leaf surfaces using chemosensilla located on the tarsi, when gustatory cues are employed. Antenna is the major olfactory organ of insects, and the various hair-like sensilla distributed on the antennae are capable of detecting the chemical odorants, such as the plant volatiles or pheromones.The Ectropis obliqua Prout(Lepidoptera: Geometridae), commonly known as tea geometrid, is a devastating defoliator of the tea plant throughout China. E. obliqua larvae are voracious caterpillars that feed on tea leaves and tender buds, causing considerable yield loss and deterioration in tea quality. Chemical control against E. obliqua is effective; however, it is undesirable due to the health and ecological risks. Alternative strategies, such as the ‘push–pull’ habitat management and synthetic sex pheromone lures, utilizing insect-behavior-modifying stimuli are developed to control this pest. E. obliqua has evolved to selectively utilize a limited number of host plants; therefore, choosing the right host plants is a formidable task based on a sophisticated sensory system. The infectation of E. obliqua dramatically increases the emission of volatiles, and the female moths are more attracted by infested tea plants and preferentially oviposited on these plants. Few studies were focused on the chemical communication between the E. obliqua and it host Camellia sinensis. Due to the poor understanding of the host-seeking mechanisms of E. obliqua, the host-selection-based control strategies against E. obliqua are not satisfactory. The deep insight of the host-selection mechanisms of E. obliqua and a better knowledge of transporting proteins and chemical receptors will lead to the discovery of target genes against this kind of pest. These benefits will lead to novel host-selection-based control stragtegy of insect pests to serve the tea industry. Using the molecular technology, we provide a preliminary study of odorant binding proteins and chemosensory recepors in E. obliqua. Besides, the ultrastructure of chemosensilla on antennae and foreleg tarsi of E. obliqua is investigated by electron microscopy. The results are shown below.1. The ultrastructure of chemosensilla on antennae and tarsi of E. obliqua To explore the putative functions of chemosensilla involved in host selection of of E. obliqua, the morphology and ultrastructure of antennal and tarsal sensilla in E. obliqua moths are investigatied by scanning and transmission electron microscopy. On antennae, sensilla trichodea(including STR I and STR II) are the most abundant sensillum type characterized by porous cuticle and dendrites in the sensillum lymph. Sensilla chaetica(SCH I–SCH III) possess a terminal pore and thick nonpo-rous wall, with dendrites in the inner lymph cavity. Three subtypes of sensilla basiconica(SBA I–SBA III) and one type of sensilla auricillica are equipped with thin porous cuticular walls and multiple dendrites in the lymph. Sensilla styloconica are equipped with two or three dendrites that lacked cuticular pores. Sensilla coeloconica possess four to seven dendrites and were double walled with spoke channels. B?hm bristles and sensilla squamiformia are also observed on the antennae. On tarsi, two subtypes of sensilla chaetica(SCH IV and SCH V) comprised the majority of chemosensilla, and are believed to function in gustatory perception. We summarize the validated functions of related sensilla, and propose potential functions of the corresponding sensilla in E. obliqua. These findings provide a working basis for investigating sensillum function and sensory mechanisms in this pest species.2. Identification and comparative study of chemosensory genes related to host selection by legs transcriptome analysis Detecting and perceiving the non-volatile chemicals of the plant surface involved in gustatory detection determines the host preference. In present study, we revealed the distribution of chemosensilla in the ventral side of female fifth tarsomere in E. obliqua. To investigate its molecular mechanism of gustatory perception, we performed Hi Seq 2500 sequencing of the male- and female- legs transcriptome and identified 24 candidate odorant binding proteins(OBPs), 21 chemosensory proteins(CSPs), 2 sensory neuron membrane proteins(SNMPs), 3 gustatory receptors(GRs) and 4 odorant receptors(ORs) genes. The RPKM value analysis revealed that 9 Eobl OBPs showed sex discrepancy in leg expression, 8 being up-regulated in female and only 1 being up-regulated in male. Eobl OBP6 has the most abundant expression in both transcriptome. These female-biased Eobl OBPs indicated an ecological adaption related with host-seeking and oviposition behavior. Eobl GR2 shares 75.3% homology with Harm GR4 which is confirmed as a fructose receptor. The tissue expression profiles of these chemosensory genes are conbstructed by q RT-PCR and RT-PCR. The majority of the Eobl OBPs are expressed in the antennae. Sixteen Eobl OBPs are specifically expressed in antennae, and 3 Eobl OBPs are highly expressed in legs. Eobl OBP7 are specifically expressed in abdomen. The other 5 Eobl OBPs are widely expressed in different tissues, while Eobl OBP5, Eobl OBP6 and Eobl OBP22 are abundant both in antennae and legs. The antennae-enriched or antennae–specific Eobl OBPs participate in the detection of sex pheromone, the location of host and the evaluation of oviposition sites. The function of the abdomen-enriched Eobl OBPs remains unclear. The expression pattern of 21 Eobl CSPs shows diverse and wide expression. Six Eobl CSPs are dominantly expressed in legs, among which Eobl CSP11 and Eobl CSP15 are highly enriched in male legs. Eobl CSP2, Eobl CSP10 and Eobl CSP16 are mostly distributed in abdomen, while Eobl CSP6 are uniquely expressed in antennae. The other Eobl CSPs are ubiquitous in most tissues. In addition, Eobl CSP1, Eobl CSP3, Eobl CSP4, Eobl CSP9, Eobl CSP12, Eobl CSP13, Eobl CSP14, Eobl CSP17, Eobl CSP19 and Eobl CSP20 are abundant in legs at a relatively high level. The function of the abdomen-enriched or leg-specific Eobl OBPs may function in gustatory sensation. Eobl SNMP1 and Eobl SNMP2 are expressed significantly higher in antennae than in other tissues of both sexes. Four ORs are mainly expresse d in the moth antennae. Among the three Eobl GRs identified, Eobl GR1 is enriched in abdomen and Eobl GR2 has antennae-enriched expression, while Eobl GR3 is detected in both legs and wings.3. Binding specificity of odorant binding protein EoblOBP3 and EoblOBP6 For the fluorescence competitive binding assay, ten tea volatiles, sixteen non-host plant volatiles, eight pest-induced volatiles, and eighteen tastants are selected as the potential ligands. Eobl OBP3 has a strong binding affinity to cis-verbenol which is repellent to E. obliqua. Eobl OBP3 also binds(Z)-3-hexenyl hexanoate which is induced by the herbivore infestation and attractive to female moths. The α-farnesene, one volatile induced by herbivore infestation, exhibits binding affinity to Eobl OBP3. Eobl OBP3 also tunes to Quercetin which belongs to the alkaloid with a taste of bitterness. Quercetin has a strong antifeeding effect on E. obliqua larvae. Eobl OBP6 has a strong binding affinity to α-farnesene and nerolidol, both of which are induced by the herbivore infestation. Besides, Eobl OBP6 can also bind benzaldehyde which is emitted from the tea shoots plucked. The terpene α-terpinene and α-caryophyllene exhibit binding affinity to Eobl OBP6. Besides, Eobl OBP6 tunes to berberine which has a strong antifeeding effect and belongs to the alkaloid with a taste of bitterness. Both Eobl OBP6 and Eobl OBP3 fail to bind the sugars and amino acids. The sensillum immunolocalization shows that Eobl OBP6 is labeled in sensilla chaetica on both tarsi and antennae.4. Identification and expression pattern of candidate olfactory receptors in E. obliqua by antennal transcriptome analysis The female- and male-antennae transcriptomes of E. obliqua adults were sequenced. Twenty-seven olfactory receptors were identified. The tissue distribution profile demonstrated that the majority of olfactory receptors are uniquely or most expressed in antennae, which indicated the participation in olfaction. Eobl OR33 was specifically expressed in male antennae. Considering the fact that Eobl OR33 is clustered with other insect pheromone receptors, it is reasonable to conclude that Eobl OR33 is a pheromone receptor of E. obliqua.