Zhao-Qun Li

Identification and Comparative Study of Chemosensory Genes Related to Host Selection by Legs Transcriptome Analysis in the Tea Geometrid Ectropis obliqua.

Host selection by female moths is fundamental to the survival of their larvae. Detecting and perceiving the non-volatile chemicals of the plant surface involved in gustatory detection determine the host preference. In many lepidopteran species, tarsal chemosensilla are sensitive to non-volatile chemicals and responsible for taste detection. The tea geometrid Ectropis obliqua is one devastating chewing pest selectively feeding on limited plants, requiring the specialized sensors to forage certain host for oviposition. 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 HiSeq 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). Several leg-specific or enriched chemosensory genes were screened by tissue expression analysis, and clustered with functionally validated genes from other moths, suggesting the potential involvement in taste sensation or other physiological processes. The RPKM value analysis revealed that 9 EoblOBPs showed sex discrepancy in the leg expression, 8 being up-regulated in female and only 1 being over expressed in male. These female-biased EoblOBPs indicated an ecological adaption related with host-seeking and oviposition behaviors. Our work will provide basic knowledge for further studies on the molecular mechanism of gustatory perception, and enlighten a host-selection-based control strategy of insect pests.

Field background odour should be taken into account when formulating a pest attractant based on plant volatiles

Attractants for pest monitoring and controlling can be developed based on plant volatiles. Previously, we showed that tea leafhopper (Empoasca onukii) preferred grapevine, peach plant, and tea plant odours to clean air. In this research, we formulated three blends with similar attractiveness to leafhoppers as peach, grapevine, and tea plant volatiles; these blends were composed of (Z)-3-hexenyl acetate, (E)-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene, benzaldehyde, and ethyl benzoate. Based on these five compounds, we developed two attractants, formula-P and formula-G. The specific component relative to tea plant volatiles in formula-P was benzaldehyde, and that in formula-G was ethyl benzoate. These two compounds played a role in attracting leafhoppers. In laboratory assays, the two attractants were more attractive than tea plant volatiles to the leafhoppers, and had a similar level of attractiveness. However, the leafhoppers were not attracted to formula-P in the field. A high concentration of benzaldehyde was detected in the background odour of the tea plantations. In laboratory tests, benzaldehyde at the field concentration was attractive to leafhoppers. Our results indicate that the field background odour can interfere with a point-releasing attractant when their components overlap, and that a successful attractant must differ from the field background odour.

Chemosensory Gene Families in Ectropis grisescens and Candidates for Detection of Type-II Sex Pheromones

Tea grey geometrid (Ectropis grisescens), a devastating chewing pest in tea plantations throughout China, produces Type-II pheromone components. Little is known about the genes encoding proteins involved in the perception of Type-II sex pheromone components. To investigate the olfaction genes involved in E. grisescens sex pheromones and plant volatiles perception, we sequenced female and male antennae transcriptomes of E. grisescens. After assembly and annotation, we identified 153 candidate chemoreception genes in E. grisescens, including 40 odorant-binding proteins (OBPs), 30 chemosensory proteins (CSPs), 59 odorant receptors (ORs), and 24 ionotropic receptors (IRs). The results of phylogenetic, qPCR, and mRNA abundance analyses suggested that three candidate pheromone-binding proteins (EgriOBP2, 3, and 25), two candidate general odorant-binding proteins (EgriOBP1 and 29), six pheromone receptors (EgriOR24, 25, 28, 31, 37, and 44), and EgriCSP8 may be involved in the detection of Type-II sex pheromone components. Functional investigation by heterologous expression in Xenopus oocytes revealed that EgriOR31 was robustly tuned to the E. grisescens sex pheromone component (Z,Z,Z)-3,6,9-octadecatriene and weakly to the other sex pheromone component (Z,Z)-3,9-6,7-epoxyoctadecadiene. Our results represent a systematic functional analysis of the molecular mechanism of olfaction perception in E. grisescens with an emphasis on gene encoding proteins involved in perception of Type-II sex pheromones, and provide information that will be relevant to other Lepidoptera species.

Identification of the genes in tea leafhopper, Empoasca onukii (Hemiptera: Cicadellidae), that encode odorant-binding proteins and chemosensory proteins using transcriptome analyses of insect heads

Empoasca onukii Matsuda is a serious pest affecting tea production throughout China. Chemosensory behaviors are important in the life cycle of E. onukii, especially for detecting plant odorants during host localizations. In this process, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate the initial biochemical recognition steps. We used a transcriptomics-based approach to identify 40 putative E. onukii OBP genes and 11 CSP genes. The encoded OBPs comprised 19 classic OBPs and 21 plus-C OBPs. A phylogenetic analysis revealed that the plus-C OBPs formed monophyletic groups, and most of the classic OBPs and CSPs were distributed among other orthologous groups. Additionally, there were significant differences between the sexes regarding the expression of five OBP genes and two CSP genes. Moreover, 36 OBP genes and six CSP genes were most highly expressed in the heads. Two CSP genes were most highly expressed in the thoraxes, while four OBP genes and three CSP genes exhibited high expression levels in the heads and thoraxes. Our data may represent a valuable resource for future functional characterizations of the E. onukii OBPs and CSPs.

Ultrastructure of Chemosensilla on Antennae and Tarsi of Ectropis obliqua (Lepidoptera: Geometridae)

Abstract Tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae) is a devastating defoliator throughout the tea plantations in China. To explore the putative functions of chemosensilla involved in host selection, the morphology and ultrastructure of antennal and tarsal sensilla in E. obliqua moths were visualized by scanning and transmission electron microscopy. Eight types (including 15 subtypes) of sensilla were identified. On antennae, sensilla trichodea (STR I and STR II) were the most abundant sensillum type characterized by porous cuticle and dendrites in the sensillum lymph. Sensilla chaetica (SCH I–SCH III) possessed a terminal pore and thick nonporous wall, with dendrites in the inner lymph cavity. Three subtypes of sensilla basiconica (SBA I–SBA III) and one type of sensilla auricillica were equipped with thin porous cuticular walls and multiple dendrites in the lymph. Sensilla styloconica were equipped with two or three dendrites that lacked cuticular pores. Sensilla coeloconica possessed four to seven dendrites and were double walled with spoke channels. Böhm bristles and sensilla squamiformia were 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.

The Odorant Binding Protein 6 Expressed in Sensilla Chaetica Displays Preferential Binding Affinity to Host Plants Volatiles in Ectropis obliqua

The monophagous tea geometrid Ectropis obliqua selectively feed on tea plants, requiring the specialized chemosensory system to forage for certain host. A deep insight into the molecular basis would accelerate the design of insect-behavior-modifying stimuli. In the present study, we focused on the odorant-binding protein 6 (EoblOBP6) with the high abundance in legs transcriptome of E. obliqua moths. qRT-PCR coupled with western blot analyses revealed the dual expression pattern of EoblOBP6 in antennae and legs. Cellular immunolocalization indicated that EoblOBP6 was predominantly labeled in the outer sensillum lymph of uniporous sensilla chaetica, which is not innervated by sensory neurons. No specific staining was observed in other sensillum types. The fluorescence competition assay showed a relatively narrow binding spectrum of recombinant EoblOBP6. EoblOBP6 could not only bind with intact tea plant volatiles benzaldehyde but also display high binding ability to nerolidol and α-farnesene which are tea plant volatiles dramatically induced by herbivore infestation. Besides, EoblOBP6 tightly bound to the aversive bitter alkaloid berberine. Taken together, EoblOBP6 displayed an unusual expression in sensilla chaetica, exhibited the potential involvement in olfaction and gustation, and may play a functional role in host location of female E. obliqua moths.

Does Background Odor in Tea Gardens Mask Attractants? Screening and Application of Attractants for Empoasca onukii Matsuda

Abstract Plant volatiles help herbivores to locate their hosts, and therefore, they could be used to help develop pesticide-free pest management strategies.To develop an attractant for tea leafhopper (Empoasca onukii), we screened nine tea plant volatile compounds for their attractiveness using Y-tube olfactometer assays. Results indicated that tea leafhoppers significantly preferred ocimene, limonene, (Z)-3-hexenol, and (Z)-3-hexenyl acetate over clean air. These compounds were combined in a blend which lost its attractiveness at concentrations below 10–2 g/ml in liquid paraffin. In field tests, the blend was attractive to leafhoppers only in autumn, but not in summer. Analyses of the tea field background odor showed that all four components of the blend were present at much higher concentrations in summer (0.05–0.001 ng/liter) than in autumn (∼10- to 25-fold lower). In fieldY-tube bioassays, compared with the tea field background odor, the blend was attractive at a concentration of 10–1 g/ml in liquid paraffin, but not at 10–2 g/ ml.These results suggest that field background odor can disrupt the attractiveness of an attractant based on plant volatiles to herbivores.

Design of an Attractant for Empoasca onukii (Hemiptera: Cicadellidae) Based on the Volatile Components of Fresh Tea Leaves

The tea leafhopper, Empoasca onukii Matsuda, is a serious pest of the tea plant. E. onukii prefers to inhabit vigorously growing tender tea leaves. The host selection of E. onukii adults may be associated with plant volatile compounds (VOCs). We sought to identify potentially attractive VOCs from tea leaves at three different ages and test the behavioral responses of E. onukii adults to synthetic VOC blends in the laboratory and field to aid in developing an E. onukii adult attractant. In darkness, the fresh or mature tea leaves of less than 1-mo old could attract more leafhoppers than the mature branches (MB) that had many older leaves (leaf age >1 mo). Volatile analysis showed that the VOC composition of the fresh leaves was the same as that of the mature leaves, but linalool and indole were not at detectable levels in VOCs from the MB. Moreover, the mass ratio differed for each common volatile in the three types of tea leaves. When under competition with volatiles from the MB, the leafhoppers showed no significant tropism to each single volatile but could be attracted by the synthetic volatile blend imitating the fresh leaves. With the removal of some volatile components, the effective synthetic volatile blend was mixed with (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, and linalool at a mass ratio of 0.6:23:12.6. These three volatiles may be the key components for the host selection of E. onukii adults and could be used as an attractant in tea gardens.

Gene Identification of Pheromone Gland Genes Involved in Type II Sex Pheromone Biosynthesis and Transportation in Female Tea Pest Ectropis grisescens

Moths can biosynthesize sex pheromones in the female sex pheromone glands (PGs) and can distinguish species-specific sex pheromones using their antennae. However, the biosynthesis and transportation mechanism for Type II sex pheromone components has rarely been documented in moths. In this study, we constructed a massive PG transcriptome database (14.72 Gb) from a moth species, Ectropis grisescens, which uses type II sex pheromones and is a major tea pest in China. We further identified putative sex pheromone biosynthesis and transportation-related unigenes: 111 cytochrome P450 monooxygenases (CYPs), 25 odorant-binding proteins (OBPs), and 20 chemosensory proteins (CSPs). Tissue expression and phylogenetic tree analyses showed that one CYP (EgriCYP341-fragment3), one OBP (EgriOBP4), and one CSP (EgriCSP10) gene displayed an enriched expression in the PGs, and that EgriOBP2, 3, and 25 are clustered in the moth pheromone-binding protein clade. We considered these our candidate genes. Our results yielded large-scale PG sequence information for further functional studies.

Evidence of Premating Isolation Between Two Sibling Moths: Ectropis grisescens and Ectropis obliqua (Lepidoptera: Geometridae).

Abstract The sex pheromones of Ectropis grisescensWarren and Ectropis obliqua Prout were both reported to contain (Z,Z,Z)-3,6,9-octadecatriene (Z3,Z6,Z9-18:H) and (Z,Z)-3,9-cis-6,7-epoxy-octadecadiene (Z3,epo6,Z9-18:H). To clarify how these two sibling geometrids maintain premating isolation, the female sex pheromones of the two species were reexamined. Gas chromatography–electroantennographic detection (GC-EAD) and gas chromatography–mass spectrometry revealed two GC-EAD-active compounds, Z3,Z6,Z9-18:H and Z3,epo6,Z9-18:H, in E. grisescens female pheromone glands as well as an additional GC-EAD-active compound, (Z,Z)-3,9-cis-6,7-epoxy-nonadecadiene (Z3,epo6,Z9-19:H), in E. obliqua female pheromone glands. Synthesized Z3,Z6,Z9-18:H and Z3,epo6,Z9-18:H elicited dose-dependent electroantennogram (EAG) responses from male antennae of both E. grisescens and E. obliqua. However, Z3,epo6,Z9-19:H only elicited dose-dependent EAG responses from E. obliqua and limited EAG responses from E. grisescens at all doses. In wind-tunnel studies, lures that contained Z3,Z6,Z9-18:H and Z3,epo6,Z9-18:H attracted E. grisescens males and had no effect on E. obliqua males.The addition of Z3,epo6,Z9-19:H to the blend of Z3,Z6,Z9-18:H and Z3,epo6,Z9-18:H strongly attracted E. obliqua males but had a limited attraction for E. grisescens males. Thus, Z3,Z6,Z9-18:H and Z3,epo6,Z9-18:H were sex pheromone components of E. grisescens, whereas Z3,Z6,Z9-18:H, Z3,epo6,Z9-18:H and Z3,epo6,Z9-19:H were sex pheromone components of E. obliqua.The presence or absence of Z3,epo6,Z9-19:H played a central role in the premating isolation of these two sibling species.