| The Tea green leafhopper Empoasca onukii Matsuda(Hemiptera: Cicadellidae)is one of the most important pests in tea plantations in China.This insect pest is widely distributed and characterized by a high fecundity.Moreover,Empoasca onukii is small,good at jumping and hiding,which makes it extremely difficult to control.For many years,Empoasca onukii has been seriously affecting the yield and quality of tea production in the main tea-producing areas in our country.The intensive study on the behavioral ecology of Empoasca onukii is an important prerequisite for the green control of the tea plant pests.The feeding behavior of the tea leafhoppers has been widely studied.Relatively,due to the lack of a rapid and accurate method for the egg detection,the research related to the oviposition behavior of this pest has still been scare,and most of the current studies have only stayed in the field investigations and analysis of the egg quantity of a few tea cultivars.This paper aimed at the Empoasca onukii,on the basis of a new method for successfully detecting their eggs on an intact tea shoot,with several technologies of insect ethology,electrophysiology,and chemical ecology were used for further surveying the biological characteristics of the oviposition by Empoasca onukii,analyzing the behavioral responses of Empoasca onukii to the visual and olfactory-related cues,and exploring the correlation between the preferential choice for oviposition by Empoasca onukii and the physical and chemical features of its host tea shoots.The main research results are as follows:1.The biological characteristics relating to the oviposition behavior of Empoasca onukii were clarifiedFirstly,the blue light detection technology was verified to quickly and accurately count the amount of the Empoasca onukii eggs on an intact tea shoot.Both the male and female leafhoppers can mate throughout the day,and the zygotes could be completed about 2 d after successful mating.According to the observations in laboratory,the gravid Empoasca onukii can lay eggs throughout the day,but the peak period is generally in the afternoon to the first of the night(17:30-23:30).The oviposition period of the female Empoasca onukii is about 20 d,with an average of 2-4 eggs laid per day and 42.8 eggs laid in total.The oviposition sites of leafhoppers on tea plants are selective,86.7% eggs are oviposited inside the tender stems and more than 50% eggs are mainly deposited at the 2nd and 3rd leaf positions.The main oviposition sites inside the stem of tea shoot are cortex and phloem where the eggs can be laid horizontally,vertically or obliquely.In the tea plantations,the oviposition of Empoasca onukii shows the relatively obvious seasonal fluctuations,the peak occurs from May to June,and there is another smaller peak in September.2.The behavioral responses of Empoasca onukii to the visual and olfactory-related cues were exploredAccording to the determinations of the oviposition behavior,the background color did not significantly affect the oviposition by Empoasca onukii when tea shoots were used as the oviposition substrate,but the different wavelengths of light and the different photoperiods showed the significant effects on the oviposition of Empoasca onukii.The treatments of the blue light(460-470 nm),the indigo light(435-445 nm)and the violet light(400-410 nm)during the peak period of egg-laying by females Empoasca onukii at night significantly inhibited their oviposition.The long light treatment can significantly stimulate the oviposition by Empoasca onukii,while the all day light or dark treatments can significantly inhibit their oviposition.The H-tube olfactory apparatus was used to test the behavioral responses of Empoasca onukii to the 10 common tea plant volatiles.The results indicated that the males Empoasca onukii had the significant olfactory response to 6 volatiles(Linalool,Nerolidol,(E)-ocimene,(E,E)-α-farnesene,(E)-2-hexenal and(Z)-3-hexenyl acetate),when the virgin females had the significant olfactory response to 7 volatiles(Linalool,Nerolidol,D-limonene,(E)-ocimene,(E,E)-α-farnesene,(E)-2-hexenal and(Z)-3-hexenyl acetate)and the gravid females had the significant olfactory response to 7 volatiles(Linalool,Nerolidol,(Z)-3-hexen-1-ol,D-limonene,(E)-ocimene,(E,E)-α-farnesene and(Z)-3-hexenyl acetate),respectively.The electroantennography technology was used for further measuring the electrophysiological responses of adults Empoasca onukii to these volatiles.The results showed that the males Empoasca onukii exhibited the significant EAG responses to 2 volatiles((E)-ocimene and(Z)-3-hexenyl acetate),when the virgin females exhibited the significant EAG responses to 2 volatiles(Linalool and(Z)-3-hexenyl acetate)and the gravid females exhibited the significant EAG responses to3 volatiles((Z)-3-hexen-1-ol,(E)-ocimene and(Z)-3-hexenyl acetate),respectively.The above conclusions indicated that the 3 tea plant volatiles,like(Z)-3-hexen-1-ol,(E)-ocimene and(Z)-3-hexenyl acetate,played an important role in the localization for oviposition by Empoasca onukii.Among them,the(Z)-3-hexen-1-ol was speculated to be a specific olfactory cue in close range localization for the ovipositing Empoasca onukii.3.The oviposition selection and the physicochemical basis of Empoasca onukii on different tea cultivars were analyzedThe oviposition selection of Empoasca onukii in the 24 widely planted tea cultivars was determined by a free selective experiment on the oviposition in laboratory and the eggs investigations in the field.The results indicated that the 8 cultivars,included Fuding White Tea,Fuyun No.6,Maoxie,Tieguanyin,Longjing 43,Banzhuyuan,Juyan Roundleaf and Huangjinya,were susceptible for Empoasca onukii oviposition,whereas the other 13 cultivars,included Jinxuan,Enbiao,Deqing Roundleaf,Zhongcha 108,Huangyan,Anji White Tea,Wuniuzao,Jiande Roundleaf,Jinguanyin,Zisun Roundleaf,Rougui,Huangmeigui and Zijuan were resistant.By investigating the egg-distributions of 24 tea cultivars in the field and measuring the shearing forces(SFs)of 24 tea cultivars at the different leaf positions in laboratory,then the relationship models between the tea-shoot tenderness and the oviposition fitness of Empoasca onukii were simulated for the 24 tea cultivars,with the oviposition fitness of Empoasca onukii represented by the number of eggs and the tea-shoot tenderness represented by the shearing force(SF)value.The results showed that the cumulative amount of Empoasca onukii eggs at the different leaf positions and the SF values were all conformed to the Gaussian cumulative distribution in the 24 tea cultivars.According to these functions,it was clear that the suitable areas for Empoasca onukii oviposition were the stem segments at the 2nd-5th leaf positions of tea shoots,the percentage of eggs was72.26 ± 1.52%,and the range of SF values was 3.00-14.25 N.The sub-suitable areas for Empoasca onukii oviposition included the leaves and the stem segment at the 1st leaf position,and the stem segments at the 6th-7th leaf positions.The percentage of eggs in the leaves and the stem segment at the 1st leaf position were 12.12 ± 1.38%,with the range of SF values 0-3.00 N,while the percentage of eggs in the stem segments at the6th-7th leaf positions was 13.45 ± 1.26%,with the range of SF values 14.25-20.96 N.However,the unsuitable areas for Empoasca onukii oviposition included all the leaves and stems that were higher than the 7th leaf position of tea shoots,and the percentage of eggs laid in this area was only 2.17 ± 0.66%,the range of SF values was greater than20.96 N.4.The physical and chemical features of tea shoots and their correlation with the oviposition selection of Empoasca onukii were revealedThe 4 Empoasca onukii oviposition-susceptible tea cultivars(Fuding White Tea,Fuyun No.6,Maoxie and Longjing 43)and 4 ovipositioon-resistant tea cultivars(Huangmeigui,Jinguanyin,Zhongcha 108 and Zijuan)were selected out for further investigations,to elaborate the correlation between several physical and chemical features of the new shoots of tea plants and the oviposition by Empoasca onukii.The results indicated that,in terms of the external morphology of tea shoots,the color of the one-bud-three-leaves in tea shoot,the length of stem internodes and the diameter of stem at the 1st-5th leaf position,and the length of trichomes on the stem at the 3rd leaf position may affect the oviposition preference of the females Empoasca onukii.In terms of the internal physical structure of tea shoots,the thickness of cortex and phloem inside the stem at the 1st-5th leaf position were the important tactile cues for Empoasca onukii to selecting the oviposition cultivars.In terms of the chemical features of tea shoots,the secondary metabolites of tea plants were analyzed by the ultra performance liquid chromatography system linked to Q-exactive orbitrap HRMS(UPLC-Q-Exactive-MS).The results showed that the contents of the lignin,the sinapyl alcohol and the γ-aminobutyric acid were gradually increased along with the growing and developing of the new shoots,while the contents of the phenylalanine,the coniferyl alcohol,the 6 catechins(C,GC,EC,EGC,ECG and EGCG)and the caffeine were gradually decreased accordingly.The contents of lignin,6 catechins and caffeine in the Empoasca onukii oviposition-resistant cultivars were generally higher than those in the susceptible cultivars,while the contents of phenylalanine and coniferyl alcohol in the susceptible cultivars were generally higher than those in the resistant cultivars.Furthermore,the results of the determinations of binary selection and non-selection for oviposition showed that the lignin,catechin metabolites and caffeine all played the deterrent roles in the process of the oviposition determination by females Empoasca onukii,while the phenylalanine and coniferyl alcohol played the stimulant roles.Finally,a comprehensive analysis was conducted for the 9 physicochemical characteristics of tea shoots that played a significant role in mediating the oviposition selection of Empoasca onukii through the above investigations.By testing the correlate distances between the influencing factors,and by comparing the differences in the measured values of each factor between the Empoasca onukii oviposition-susceptible and resistant cultivars,it was clear that the 4 physicochemical features of tea shoots(catechin,lignin,the length of stem trichome and the stem diameter)played the most important role in mediating the oviposition decision of Empoasca onukii.This study focused on the oviposition behavior of Empoasca onukii.The biological characteristics associated with oviposition by Empoasca onukii were investigated.The orientation cues to the oviposition on the tea shoots by Empoasca onukii were clarified.A number of tea cultivars were screened and identified for the susceptibility or resistance to oviposition by Empoasca onukii.Furthermore,the mediated effects of tea shoot relating with the physical and chemical features on the oviposition preference for Empoasca onukii were further revealed.The conclusion of this study provided an important theoretical basis for further understanding the mechanisms of the localization and selection for Empoasca onukii oviposition,and for developing the green control technology of tea leafhoppers based on the regulation of oviposition behavior in the future. |
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