Introduction

Rice is an essential food crop in China1,2but the brown planthopper, Nilaparvata lugens (Stål), is a pest that poses a tremendous threat to rice. It can lead to major reductions in rice yields due to hopperburn, causing substantial economic losses3,4. At present, the control of N. lugens is mainly reliant on chemical pesticides such as pymetrozine5dinotefuran6nitenpyram7and triflumezopyrim8. Requirements for the quality and yield of agricultural products are steadily increasing with the enhancement of people’s awareness of agricultural product safety and changes in their concepts of consumption9. Consequently, the pursuit of environmentally friendly prevention and control is a potential future development trend. Moreover, with the implementation of pesticide reduction, plant-derived pesticides have received greater attention due to their low toxicity and minimal residue10,11.

Plant essential oils generate a wide range of biological activity against pests in a variety of ways12. Their repellent activity has been widely applied in the prevention and control of crop pests, sanitary pests, and stored grain pests13,14,15. For instance, in terms of crop pests, Origanum majorana(L.) essential oil has an excellent repellent effect on Myzus persicae (Sulzer), and Aphis fabae Scopoli (Hemiptera: Aphididae)16. Additionally, Foeniculum vulgare (Mill.) essential oil and Tagetes species essential oil exert toxic and repellent activity toward adults and nymphs of Diaphorina citri Kuwayama (Hemiptera: Liviidae)17. Several essential oils have been registered as botanical mosquito repellents, including Citrus (L.), citronella Cymbopogon citratus L. Rendle, Mosla chinensis Maxim oil, lemon amine Eucalyptus citriodora Hook. f., and cat mint (Nepeta cataria L.). Besides, the repellency of neem Azadirachta indica (A. Juss.), yellow-flowered oleander Thevetia peruviana (Pers.), and red eucalyptus, Eucalyptus camaldulensis (Dehnh.) toward Drosophila melanogaster Meigen adults are 91.44%, 72.19%, and 72.80%, respectively18. Regarding stored grain pests, plant essential oils have been used in control and prevention in numerous successful studies. The Clerodendrum bungei essential oil has been found to have repellent activity against Tribolium castaneum (Herbst), Lasioderma serricorne (F.), and Liposcelis bostrychophila Badonnel through the filter paper method19. Moreover, the essential oils of Spike lavender, Lavandula spica Medik. (Lamiaceae), Tripterygium wilfordii Hook.f. fennel, Artemisia argyi (H.Lév.), Cinnamon Cinnamomum cassia (L.) D. Don, citronella, and clove Syringa oblata Lindl. also have strong repellent effects on Tribolium castaneum20,21. It has been reported that the mechanism of plant-derived repellents involves interaction between volatile compound odor molecules and insect olfactory receptors, thereby inducing insect repellent behavior. For thousands of years, pyrethrum extract has been widely used around the world as an insect repellent. Previous studies have established that the repellent effect of pyrethrum extract is principally related to olfactory perception22,23,24,25. For example, D. melanogaster was used as the model for a study on the insect olfactory system26. The results of the study revealed that the DmelOR7a and DmelOR59b genes were involved in the process of perceiving pyrethrins. Besides, DmelOR98a is involved in perceiving farnesene, a secondary metabolite of pyrethrins. Additionally, the (E)-β-farnesene in pyrethrins has been found to activate Aedes aegypti (L.) sbt-1 A neurons and olfactory receptors (AgOr31), while the repellent effect of pyrethrins on A. aegypti is weakened by the knockdown of AgOr3127. AgamOBP4 was involved in the recognition and transmission of citronellal by mosquitoes28. PopeOBP16 involved in odor recognition in the potato tuber moth, Phthorimaea operculella (Zeller) of adults, had the strongest binding affinity with the plant volatiles29. It was also found that NlOBP8 plays an important role in the recognition of the volatile linalool by brown planthoppers30. All these studies demonstrate the important roles of OBPs in olfaction. Olfaction in these three planthoppers plays key roles in the seeking of rice plants, as volatiles and extracts of rice plants can trigger a strong attraction response. However, functional studies to determine their olfactory mechanism have not been performed62. It was therefore particularly important to interfere with host recognition by brown planthoppers. Therefore, we decided to ascertain the repellent activity of 16 essential oils against N. lugens. Subsequently, we examined the repellent, antifeedant, and oviposition effects of citronella essential oil and its principal constituents geraniol and citronellal on N. lugens. Furthermore, differences in expression after treatment with geraniol on odorant-binding proteins (OBPs) in N. lugens were also evaluated through dsRNA microinjection technology. The aim of this paper is to screen out compounds with outstanding repellent activity against N. lugens, to investigate the repellent mechanism, and to provide a theoretical basis for the synthesis of new repellent agents.

Results

Repellency of plant essential oils to N. lugens

Figure 1 reveals that the repellent activity of the plant essential oils was positively related to their concentrations. The results of the H-tube olfactometer assays indicated that the essential oils of citronella, lemon citronella Cymbopogon citratus (DC.) Stapf, nutmeg Myristica fragrans (Houtt.), cinnamon, Litsea cubeba (Lour.) Pers.[L. citrata Blume], holly Ilex chinensis, clove Syringa oblata Lindl., star anise Illicium verum Hook. and perilla Perilla frutescens (L.) Britt. exhibited significant repellent activity on nymphs at a concentration of 5 µg/cm2, 50 µg/cm2 and 500 µg/cm2. Additionally, Rosemary Rosmarinus officinalis (L.) and patchouli Pogostemon cablin (Blanco) Benth. had effective repellent activity on nymphs at 50 and 500 µg/cm2 (Fig. 1B, C). Prickly ash Zanthoxylum bungeanum Maxim. and houttuynia cordata Houttuynia cordata Thunb. had effective repellent activity on nymphs only at 500 µg/cm2 (Fig. 1C). However, the pepper Piper nigrum L., garlic Allium sativum L. and evodia Tetradium ruticarpum (A. Juss.) T. G. Hartley essential oils showed no repellence or attractant effect on nymphs. Therefore, citronella, lemon citronella, nutmeg, cinnamon, Litsea cubeba, holly, clove, star anise and perilla were selected for the ensuing bioassay that measured adult response.

Fig. 1
figure 1

H-tube olfactometer bioassay of nymphs. The behavioral responses of nymphs to 16 plant essential oils screened above at (A) 5 µg/cm2, (B) 50 µg/cm2, (C) 500 µg/cm2 concentration, respectively. The white bar represents plant essential oils, and the black bar represents different hexane (control) P values were determined by the χ-square test. Ns, no significant difference (P > 0.05); *, significant difference (P < 0.05); **, highly significant difference (P < 0.001 or P < 0.0001).

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Among the nine essential oils, citronella had the best repellent effect on adults (Fig. 2). Citronella essential oil at 5 µg/cm2 showed high selectivity for n-hexane (χ2 = 37.90, P < 0.0001) (Fig. 2A) repellency was 83.33% (Table S1). Moreover, at 50 µg/cm2 (Fig. 2B), the selectivity of the hexane rose to 89.23%, considerably higher than the treated (10.76%) (χ2 = 51.38, P < 0.0001) repellency was 84.09% (Table S1). Finally, the selectivity of the hexane at 500 µg/cm2 was 94.20% (χ2 = 70.63, P < 0.0001) (Fig. 2C), repellency was 91.43% (Table S1). Besides, holly oil, Litsea cubeba, cinnamon, nutmeg and lemon citronella essential oil also repellent effect on adults. Furthermore, adults showed a significantly repellent response to perilla (χ2 = 10.18, P = 0.0014 and χ2 = 18.59, P = 0.0014), star anise(χ2 = 16.00, P < 0.0001, χ2 = 40.23, P < 0.0001) and clove (χ2 = 16.00, P < 0.0001, χ2 = 25.00, P < 0.0001) essential oil only at 50 µg/cm2 and 500 µg/cm2 (Fig. 2B, C).

Fig. 2
figure 2

H-tube olfactometer bioassay of adults. The behavioral responses of adults to 9 plant essential oils screened above at 5 µg/cm2, 50 µg/cm2, 500 µg/cm2 concentration, respectively. P values were determined by the χ-square test. Ns, no significant difference (P > 0.05); *, significant difference (P < 0.05); **, highly significant difference (P < 0.001 or P < 0.0001).

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Figure 3 indicates that geraniol and citronellal (Fig. 3A) showed a substantial repellent effect on adults. Geraniol at 5 µg/cm2, 50 µg/cm2, and 500 µg/cm2 had a substantial repellent impact on adults, with a selectivity (33.33%, 12.31%, and 8.57%)(χ2 = 7.114, P = 0.0076; χ2 = 46.96, P < 0.0001; χ2 = 61.22, P < 0.0001) much be belower than the hexane (66.67%, 87.69%, and 91.43%), repellency were 34.62%, 85.40%, and 91.76%, respectively(Table S1). However, under the treatment of citronellal at at 5 µg/cm2, the selectivity of N. lugens to hexane was 71.56%, which was superior to that of geraniol (66.67%) (Fig. 3A, B), repellency were 43.22%(Table S1). At medium and high concentrations, repellency were 49.74% and 64.09% were below those of geraniol (85.40% at 50 µg/cm2, 91.76% at 500 µg/cm2) (Table S1). The results suggested that geraniol had more powerful repellent activity than citronellal.

Fig. 3
figure 3

H-tube olfactometer bioassay of adults. The behavioral responses of adults to 2 plant essential oils screened above at 5 µg/cm2, 50 µg/cm2, 500 µg/cm2 concentration, respectively. P values were determined by the χ-square test. Ns, no significant difference (P > 0.05); *, significant difference (P < 0.05); **, highly significant difference (P < 0.001 or P < 0.0001).

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Selective feeding deterrency of geraniol and citronellal against N. lugens

As Fig. 4 reveals, N. lugens preferred to feed on normal rice. Both geraniol and citronellal showed good feeding deterrency activity against N. lugens wakame in the concentration range of 4–400 µg. At all three dose treatments, geraniol and citronellal treatment for 12 h, BPH take on blank rice was significantly higher than the treatment. After 12 h of treatment with 4 µg geraniol, 12.00% (Fig. 4A) of N. lugens feeding on treated rice, which was significantly lower than that of the control (78.67%) (P < 0.0001). Meanwhile, After 12 h of treatment with 4 µg citronellal, 13.33% (Fig. 4D) of N. lugens feeding on treated rice, which was significantly lower than that of the control (76.67%; Fig. 4D) (P = 0.0079). Besides, After 12 h of treatment with 40 µg geraniol, only 10.67% N. lugens feeding on treated rice, which was significantly lower than the (85.33%; Fig. 4B) (P = 0.0079) in the control. After 12 h of treatment with 40 µg citronellal, only 9.33% N. lugens feeding on treated rice, which was considerably lower than the (69.33%; Fig. 4E) (P = 0.0079) in the control. Furthermore, After 12 h of treatment with 400 µg geraniol, only 1.33% N. lugens feeding on treated rice, which was considerably lower than the (94.67%; Fig.4C) (P = 0.0079) in the control. After 12 h of treatment with 400 µg citronellal, 16.00% N. lugens feeding on treated rice, which was considerably lower than the (82.67%; Fig. 4F) (P < 0.0001) in the control.

Fig. 4
figure 4

Feeding deterrency of geraniol and citronellal against 5th instar nymph, (A–C) is geraniol treatment, (D–F) is citronellal treatment on rice under different concentrations. Data was analyzed by student t-test. *P < 0.05, **P < 0.01.

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Oviposition deterrence of geraniol and citronellal against N. lugens

Oviposition deterrence assays revealed that geraniol and citronellal exhibited variable oviposition deterrence against adult female N. lugens. The number of eggs laid by N. lugens gradually fell with increasing geraniol concentrations, and was significantly lower than the control group. Different concentrations of citronellal treatment also had a similar effect. At a concentration of 20 mg/L, the oviposition deterrence rate of geraniol and citronellal on female adults were 56.28% and 46.67%, respectively (Fig. 5A). Moreover, the oviposition deterrence rate of geraniol and citronellal at a concentration of 200 mg/L were 68.36% and 66.33% (Fig. 5B). At the highest concentration, both substances had the strongest oviposition deterrence activity, but geraniol was more effective than citronellal (83.95% vs. 79.15%; Fig. 5C). Therefore, geraniol exhibited greater oviposition deterrence toward adult female N. lugens than citronellal.

Fig. 5
figure 5

Oviposition deterrence (mean + sem, n = 10) of N. lugens by geraniol and citronellal. (A), (B) and (C) represent three different concentrations of 20 mg/L, 200 mg/L and 2000 mg/L respectively. Data was analyzed by student t-test. *P < 0.05, **P < 0.01.

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No selective feeding deterrency of geraniol and citronellal against N. lugens

As Fig. 6 shows, feeding deterrent activity was calculated using the landing numbers of N. lugens after treating rice seedlings with different doses of geraniol or citronellal. Treatment with 40–400 µg geraniol (Fig. 6A) had significant feeding deterrent activity on the insects at 1 h, 2 h, and 4 h. However, the non-selective feeding deterrent activity fell over time. The on selective feeding deterrent activity on N. lugens was strongest at 1 h and 2 h after treatment with both dosages of citronellal (Fig. 6B), but the feeding deterrent activity was not significant at 4 h. After geraniol and citronellal treatment, the number of brown planthoppers on the rice seedlings was the lowest at 1 h, then the number increased over longer periods (Fig. 6). The median antifeeding concentration (AFC50) value of geraniol and citronellal against the third-instar nymphs were 0.23 mg/mL and 0.72 mg/mL, respectively (Table 1). The results also indicated that geraniol exerted more intense feeding deterrent activity than citronellal31,32,33.

Fig. 6
figure 6

Deterrency in a no-choice test of geraniol and citronellal against 5th instar nymphs. (A: geraniol B: citronellal) Different lowercase letters on the histogram indicate significant differences at the level of P < 0.05, according to Tukey’s multiple comparison test.

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Table 1 Deterrency in a no-choice test of citronellal and geraniol against nymphs.
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Expression of odorant binding protein

In order to determine the specific OBPs in response to geraniol, we identified several potential OBPs in N. lugens by RT-qPCR. The qPCR findings showed that the relative expression levels of five olfactory genes in the geraniol treatment groups were significantly down-regulated. Compared to the control group, the relative expression levels of NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 were down-regulated by 44.40% (P = 0.0011), 51.32% (P < 0.001), 30.16% (P = 0.013), 53.89% (P < 0.001), and 44.26% (P = 0.006), respectively. However, the expression levels of NlugOBP5, NlugOBP6, NlugOBP7, NlugOBP8, and NlugOBP10 did not change significantly after geraniol treatment (Fig. 7). This suggests that the expression levels of five odorant-binding proteins of N. lugens were affected by geraniol. We speculate that NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4 and NlugOBP9 may be activated and involved in regulating the recognition of geraniol by N. lugens34,35,36.

Fig. 7
figure 7

Relative expression levels of NlugOBPs in N. lugens after exposure to geraniol. Data was analyzed by student t-test. *P < 0.05, **P < 0.01.

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RNAi and olfactory behavior of N. lugens

To investigate the role of NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 in the recognition of geraniol by OBPs, RNAi technology was used to silence these genes. The qRT-PCR results showed that the relative expression levels of the relevant OBP genes in N. lugens all decreased by more than 84.79% after 48 h of silencing, compared with dsGFP. Moreover, they all fell by more than 96.21% after 72 h (Fig. 8A, C, E, G). This indicated that RNAi effectively silenced the expression of the target genes NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9. A selective experiment was conducted to test the effect of RNAi on geraniol recognition by N. lugens (Fig. 8B, D, F, H, J). The repellent activity of individuals injected with dsOBP1 was attenuated by only 17.00% (Table S2), while the selectivity of the non-injection and dsGFP control group remained high, at 76.36% (χ2 = 4.56, P < 0.01) and 75.47% (χ2 = 4.58, P < 0.01) (Fig. 8B) repellency were 54.02%, and 53.87%, respectively (Table S2). In stark contrast, the repellent activity of dsOBP2, dsOBP3, dsOBP4, and dsOBP9 (Fig. 8D, F, H, J) did not change significantly, compared with the non-injection and dsGFP groups. Therefore, NlugOBP1 may be involved in the perception of geraniol.

Fig. 8
figure 8

RNAi and olfactory behavior of N. lugens. (A,C,E,J,I,K) Interference efficiency of the OBPs genes in N. lugens. (B,D,E,H,L) Analysis of the H-tube olfactory choice assay to geraniol and hexane by NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 gene knocked-down N. lugens. The dsGFP refers to the control group fed with double-stranded RNA sequences of the green fluorescent protein gene. The NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 refers to the NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 gene knocked-down experimental group. (A,C,E,J,I,K) The data are shown as the mean ± sem, N = 3. Data were statistically analyzed by one-way ANOVA, followed by Tukey’s test. (B,D,E,H,L) The data were analyzed using a Chi-square (χ2) test. *P < 0.05, **P < 0.001 or P < 0.0001, and ns represents no significant difference, N = 150.

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Discussion

Repellent activity is a crucial index for evaluating the effectiveness of a substance37,38. In this paper, we evaluated the repellent activity of 16 kinds of plant essential oils against N. lugens. It was discovered that the repellent effect of citronella essential oil was excellent, and was similar to its activity against Tetranychus pueraricola (Acari: Tetranychidae)39Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)40Tribolium castaneum41and A. aegypti42. Specifically, the repellent activity of geraniol and citronellal, two primary constituents of citronella, were higher, with repellency of 91.78% and 64.09%, respectively. However, the activity of citronellal toward N. lugens and the tobacco beetle L. serricorne. is different, since citronellal enhances the strength of attraction toward tobacco beetle43. These differences in results may be due to the monomeric compounds of essential oils acting as attractants or having negligible effects at lower concentrations. At higher concentrations, they often exhibit repellant effects9,44.

From an ecological perspective, antifeedants are crucial because they do not kill the target insects directly, instead leaving them susceptible to their natural predators and assisting in preserving the ecological balance45,46. In this study, the feeding deterrency of geraniol and citronellal toward nymphs AFC50 were 0.23 mg/mL and 0.72 mg/mL at a concentration of 2000 mg/L, and there was a certain concentration dependence. The feeding deterrent activity of citronella essential oil against Spodoptera litura (F.) corresponds closely with its effect on N. lugens47but its antifeedant activity against Mythimna separata (Walker) (Lepidoptera: Noctuidae) is quite different41. This may be explained by the varying sensitivities of the chemical receptors of different insects toward plant essential oils48,49,50. Experiments have been conducted to demonstrate that rice dwarf virus (RDV)-infected N. lugens stinging rice led to changes in rice volatiles51and some plant volatiles attracted N. lugens, enhanced host localization, and accelerated the spread of rice viruses, side by side with the fact that reducing brown planthopper colonization on rice reduces the incidence of virus disease. The same was observed in Bemisia tabaci and aphids, where a reduction in insect population rates reduced the incidence of the virus52,53,54. These demonstrate that virus transmission can be reduced by reducing the population of phytophagous insects.

Oviposition deterrence directly affect the survival and reproduction of insects by interfering with their selection of host plants. Thus, they are considered to be an important tool for controlling the number of pests55. Dilly et al.56 reported similar results, whereby citronella essential oil repelled female A. aegypti adults and controlled the number of mosquitoes. In this paper, we discovered that the oviposition deterrence of geraniol and citronellal, the main components of citronella essential oil, toward adult female N. lugens were 83.95% and 79.15%, respectively. However, a previous study noted that only 500 mg/L geraniol inhibited the hatching of 70% of root-knot nematode (Meloidogyne spp.) eggs46. The reason for this dissimilarity in results may be because root-knot nematodes are more sensitive to citronella than N. lugens57,58.

The peripheral system of insects is mainly responsible for the selective and efficient detection of substances in the environment. The peripheral system of insects is mainly responsible for the selective and efficient detection of compounds from the environment, which is composed of olfactory functional proteins such as OBPs, chemosensory proteins (CSPs), odorant receptors (Ors) and so on59,60,61. In particular, the OBPs in insect antennal lymph play a vital role in insect olfactory perception and other life processes. Moreover, they are strongly involved in the insect recognition process of information substances62,63. When insects are exposed to certain chemicals, the expression of the peripheral olfactory genes is regulated to promote behavioral plasticity. The expression of NlugOBP1, NlugOBP2, NlugOBP3, NlugOBP4, and NlugOBP9 was down-regulated after geraniol treatment. To verify this hypothesis, we silenced the four odorant-binding proteins by injecting dsRNA and found that only the silenced NlugOBP1 did not exhibit significant chemotaxis after geraniol treatment. Therefore, NlugOBP1 may be the pivotal odorant-binding protein that is involved in the perception of geraniol by N. lugens, thereby playing a crucial role in olfactory selection. In the future, the mechanism of geraniol disrupting olfactory recognition and interfering with feeding in brown planthopper can be further clarified by western blot and protein purification and competitive fluorescence binding assay of NlugOBP1.

Previous studies established that the highly expressed odorant-binding proteins NlugOBP364, NlugOBP865, NlOBP830, and OBP79764, found in the antennae of N. lugens and SfurOBP11 and LstrOBP2 in white-backed planthoppers Sogatella furcifera (Horváth) and small brown planthopper Laodelphax striatellus (Fallén) (small brown planthopper) were all involved in the recognition of host volatiles, which concurs with the results in this paper. NlugOBP330 participates in the identification of hosts in N. lugens and is also possibly related to their growth and development. Interestingly, NlOBP3, which was detected in the abdomen of N. lugens, is involved in the resistance of brown planthoppers toward nitenpyram and sulfoxaflor66. These results may be due to the different functions of the OBPs expressed in various tissue types. A previous study reported that geraniol derivative as a lead compound had satisfactory repellent activity (51.4%) and high binding affinity with pea root ApisOBP967. However, only this study examined the binding affinity between geraniol and NlugOBP1 of N. lugens. Since OBPs bridge the interaction between odorants and Ors, onfirming the function of NlugOBP1 in N. lugens will benefit further studies concerning the interactions between odorants and ORs68. The findings of this study provide further insights into olfactory plasticity in related insect species. We will also analyze the expression of Ors gene in geraniol, conduct molecular docking test between geraniol and NlugOBP1 and Ors, and the effect of RNAi silencing of Ors on the avoidance activity of N. lugens, so as to elucidate the avoidance mechanism of geraniol on N. lugens29.

The results of this study indicated that geraniol repels N. lugens and affects its feeding, egg-laying, and other related behaviors. Further investigation revealed that NlugOBP1 may be involved in the olfactory behavior perception of brown planthoppers. These findings provided preliminary information for the development of essential oils and plant compounds such as geraniol as repellence agents. This study also introduced a novel pest control approach that is both environmentally friendly and sustainable.

Materials and methods

Insects and reagents

The N. lugens samples used in this study were originally collected from rice fields in Huishui County, Guizhou Province, China, in 2022. The rice seeds collected in this study has been licensed by farmers in Huishui County. The insects and rice plants were maintained in a climate chamber at 27 ± 1℃, with a relative humidity between 70% and 80% and a light/dark photoperiod of 16 h/8 h. The following items were purchased from Ji’an Bolui Flavor Oil Co., Ltd. (China): Garlic essential oil, Patchouli essential oil, Prickly ash essential oil, Rosemary essential oil, Evodia essential oil, Houttuynia cordata essential oil, Perilla essential oil, Star anise essential oil, Holly oil, oil, Cinnamon essential oil, Nutmeg essential oil, and Lemon citronella essential oil. Additionally, Pepperessential oil, Clove oil, Citronella essential oil, Geraniol, and Citronellal were supplied by Shanghai Macklin Biochemical Co., Ltd.

Repellency activities

An H-tube Fig. 9 olfactometer consisting of two vertical tubes (4 cm diameter, 27 cm height) and a transverse tube (3 cm diameter, 31 cm height) was used to test the behavioral responses of BPHs to odorants. 16 types of essential oil were independently dissolved in hexane to produce test solutions at concentrations of 20 mg/L, 200 mg/L, and 2000 mg/L. Hexane was used as the solvent since previous experiments confirmed that it has no repellent effect on N. lugens. In the selective experiment using the H-type olfactometer, 200 µL of plant essential oil was dripped onto cotton swabs, which were placed at the bottom of the tube. Meanwhile, cotton swabs with 200 µL hexane were placed on the control side. Fifteen newborn BPH adults were carefully placed in the center of the transverse tubes which were sealed with gauze at both ends. After one hour, the number of N. lugens within 9 cm from the ends of the transverse tubes was recorded and repeated 10 times. The experimental method for the nymphs was the same as above, except that the 15 adults were replaced by 15 nymphs. Subsequently, the percentage of repellency (PR) was calculated as follows29,43:

$$~~Percentage~repellency~ = \frac{{N_{C} - N_{t} }}{{N_{C} + N_{t} }} \times 100$$
(1)

.

Where NC represents the number of insects in control and Nt represents the number of insects in treatment.

Fig. 9
figure 9

Schematic diagram of H-type olfactory system.

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Selective feeding deterrent activities

Following Kang’s experimental method69,70two rice seedlings at tillering stage were placed in plastic cups with hydroponic nutrient solution and covered with glass tubes with holes (40 mm diameter with 24 holes) at the stalks of both seedlings, sealed with seaweed at both ends. One seedling stem was smeared with 200 µL (20 mg/L, 200 mg/L, 2000 mg/L) plant essential oil monomer compound using a brush, while the other was smeared with 200 µL hexane as a control. Then, 15 nymphs were placed in a temperature-controlled (25 ± 1℃) and light-controlled (L: D = 16:8) greenhouse. The number of nymphs on each seedling was observed and recorded after 1 h, 2 h, 4 h, 12 h, and 24 h, and each treatment was repeated ten times.

Oviposition deterrence

The bioassay method followed the same procedure as the antifeedant activity experiment, except that the 15 nymphs were replaced by five pairs of adults. The number of egg mass of female N. lugens and the total number of eggs in each rice plant were investigated at 96 h. The oviposition deterrence was calculated using Eq. (2).

$$\:\text{O}\text{v}\text{i}\text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{o}\text{n}\:\text{d}\text{e}\text{t}\text{e}\text{r}\text{r}\text{e}\text{n}\text{c}\text{e}\:\left(\%\right)=\frac{Nc-Nt}{Nc+Nt}\times\:100$$
(2)

.

where Nc is represents the total number of eggs on control rice and Nt indicates the total number of eggs on treated rice.

Feeding deterrency in a no-choice test

Two rice seedlings at tillering stage were placed in plastic cups with hydroponic nutrient solution and covered with glass tubes with holes (40 mm diameter with 24 holes) at the stalks of both seedlings, sealed with seaweed at both ends. Using a brush, the seedlings were smeared with 200 µL hexane soluble plant essential oil monomer compound. After 30 min, 15 fourth-instar nymphs were placed in a temperature-controlled (25 ± 1℃) and light-controlled (L: D = 16:8) greenhouse. The number of nymphs on each seedling was observed and recorded after 1 h, 2 h, 4 h, 12 h, and 24 h, and each treatment was repeated ten times. The feeding deterrency of the plant essential oil on nymphs were calculated using the following equation:

$$\:\:\:\:\text{F}\text{e}\text{e}\text{d}\text{i}\text{n}\text{g}\:\text{d}\text{e}\text{t}\text{e}\text{r}\text{r}\text{e}\text{n}\text{c}\text{y}\:\:\left(\%\right)=\frac{Nc-Nt}{Nc+{N}_{t}}\:\times\:100$$
(3)

.

where Nc represents the number of nymphs in the control rice and Nt is the number of nymphs in the treated rice.

qRT-PCR validation of mRNA expression in OBPs

The specific olfactory genes that encoded the olfactory proteins were screened using the approach devised by Ma and Zhou71. After treatment using the repellent activity method, N. lugens specimens with chemotaxis activity were selected and fifth-instar nymphs were collected from the treatment and control groups. Total RNA was extracted using the MolPure® TRIeasy Plus Total RNA Kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA was reverse-transcribed from total RNA using HiScript II Q RT SuperMix (#R212-01, Vazyme). Subsequently, qRT-PCR was conducted on a Roche Light Cycler® 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany) using the SYBR Green Supermix Kit (#11202ES08, Yeasen). The guanine-nitrogen (7) methyltransferase gene (N118S) was used as an internal control to quantify the OBP level. The relative expression level was computed using the 2−∆∆Ct method. Three independent biological replicates and three technical replicates were performed.

Microinjection and repellency analysis

The dsRNA templates were produced using PCR with primers containing the T7 promoter sequence. The PCR products and dsRNA were synthesized and purified using a MEGAscript T7 High-Yield Transcription Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. A 431 bp dsDNA encoding green fluorescent protein (GFP) was synthesized and used as a negative control for RNAi experiments72. All primers for qPCR and dsRNA synthesis are listed in Table 2. Fifth-instar nymphs were anesthetized with carbon dioxide for 10 to 20 s. Subsequently, 50 nL of purified dsRNA encoding OBP or GFP was injected directly into the thorax between the prothorax and the mesothorax. After injection, the brown planthopper was reared on rice seedlings at the 4 to 5-leaf stage. The insects from each treatment group were collected at 48 h and 72 h after injection to test the interference efficiency and perform bioassays. To assess the sensory behavior toward geraniol after exposure to OBP RNAi, N. lugens were collected 48 h after injection to undergo an avoidance activity assay. A two-choice bioassay was conducted to test the behavioral response of N. lugens using an H-tube olfactometer. The olfactometer was the same as that used in a previous study. Each treatment and control was replicated four times with 20 individuals per replicate65.

Table 2 Primers used in qRT-PCR and DsRNA synthesis.
Full size table

Data analysis

Statistical analyses were conducted using Microsoft Excel 2019. All data were checked for normality and equality of variances prior to statistical analysis. Data from two groups were analyzed by paired or independent Student’s t test when data were in a normal distribution. A Chi-square goodness of fit test was used for the BPHs preference assay data. In bar plots, the data is presented as mean ± sem. Three or more treatments were analyzed using one-way anova followed by tukey’s hsd post-hoc tests.GraphPad Prism 8.0 (GraphPad Software Inc., USA) software is used for chart drawing and combination.