Host-Plant Volatiles Enhance the Attraction of Cnaphalocrocis medicals (Lepidoptera: Crambidae) to Sex Pheromone

Cnaphalocrocis Guenée (Lepidoptera: Pyralidae) is a notorious pest of rice, Oryza sativa L.(Poaceae). Sex pheromone and host-plant volatiles can trap C. medinalis separately. To improve the trap eciency of sex pheromone, we rst tested the synergistic effect of 8 host-plant volatiles, including 2-Phenylethanol, 1-Hexanol, 1-Heptanol, (Z)-3-Hexenal, (E)-2-Hexenal, Octanal, Valeraldehyde, and Methyl Salicylate on the attraction of C. medinalis to the female-produced sex pheromone in electroantennography. The addition of (E)-2-Hexenal, Methyl Salicylate, Valeraldehyde, and (Z)-3-Hexenal increased electroantennogram response of C. medinalis to sex pheromone. Further testing of the mixtures of these four compounds and sex pheromone in wind tunnel experiments indicated that additive (E)-2-Hexenal or Methyl Salicylate stimulated the landing behaviors of both male and female C. medinalis compared with sex pheromone alone. Field evaluations showed that mixtures of sex pheromone and (E)-2-Hexenal or Methyl Salicylate resulted in signicantly higher catches to male moths than sex pheromone alone. Using 1:1 and 1:10 combinations of the sex pheromone and (E)-2-Hexenal, showed a synergistic effect of 95% and 110%, respectively. Furthermore, 1:1 and 1:10 mixtures of the sex pheromone and Methyl Salicylate exhibited a synergistic effect of 69% and 146%, respectively. These results may provide the basis for developing ecient pest management strategies against C. medinalis using host-plant volatiles and insect sex pheromones. (1-NPN) as a uorescent probe demonstrated that trans-11-tetradecen-1-yl acetate, the sex pheromone component of Loxostege sticticalis, and (E)-2-Hexenal, the most abundant plant volatiles in essential oils extracted from host plants, had high binding anities to LstiGOBP2 (Yin et al. 2012). Grapholita molesta OBP12 (GmolOBP12) displayed a higher expression level in male antennae than in female


Introduction
Most insects use odors present in the external environment released by conspeci cs, prey, natural enemies, or host plants to nd food, communicate with their mates, and identify suitable oviposition sites (Badeke et al. 2016;Bruce and Pickett 2011;Dupuy et al. 2017). Insect sex pheromones are chemical substances used for intraspeci c identi cation and communication (Badeke et al. 2016). So far, the majority of moth sex pheromones identi ed are released by females used e ciently to attract the males and locate mate sites (Roelofs et al. 2002). The control of moths in the eld through synthetic sex pheromone blend is feasible by population monitoring, mating disruption, and mass trapping (Mayer 2019). However, sex pheromones mixed up with other volatile compounds under natural conditions, including those released by surrounding vegetation. If insects in natural settings recognize signals from food or plants from which biologically active odorants emanate, human-made lures based solely on sex pheromone are not likely to be competitive when placed in the eld (Deng et al. 2004a;Kawazu et al. 2000).
Host-plant volatiles are low molecular weight, lipophilic character, and high volatility compounds (Loreto and Schnitzler 2010;Pichersky et al. 2006) full of natural environments (Dicke et al. 2009;Pinto et al. 2008). In natural conditions, host-plant volatiles plays an essential role in guiding phytophagous insects to feeding, mating, and oviposition sites (Von Arx et al. 2012). Increasing evidence suggests that host-plant volatiles and insect sex pheromone are interactive under eld conditions (Reddy and Guerrero 2004). Certain moths acquire or sequester host-plant compounds and use them as a sex pheromone, sex pheromone precursors, or particular host plant cues for producing or releasing sex pheromone (Landolt and Phillips 1997;McNeil and Delisle 1989;Nishida 2014). It has been con rmed that host-plant volatiles enhances sex pheromone responses in some species of Lepidopterans moths. One eld test showed that a mixture of Holotrichia parallela sex pheromone and (E)-2-hexenyl acetate could increase sex pheromone baited trap captures in the eld, thereby improving current uses of sex pheromones (Ju et al. 2017). E-β-ocimene and E-β-farnesene, two of the prominent volatiles of certain Grapholita molesta hosts, signi cantly raised the capture of G. molesta male moths than traps baited only with sex pheromone in the eld (Xiang et al. 2019). An e cient sex pheromone attractant is still missing for the management of Ectomyelois ceratoniae because the major pheromone component is unstable. Interestingly, the combination of female sex pheromone and volatiles from cracked pomegranates have great potential for application in baited trap capture of carob moth (Hosseini et al. 2017). Host-plant volatiles combined with female sex pheromone, have broad application prospects in integrated pest management (IPM) strategies.
Subsequently, we conducted wind tunnel and eld tests to determine if these compounds enhance sexual communication in C. medinalis. The main objective is to facilitate the development of more e cient lures for monitoring and controlling this insect pest.

Materials And Methods
Insects. C. medinalis used in this study came from an experimental plot in Yangzhou University, Yangzhou,China (119° 43 E;32° 39 N). Larvae were reared on rice seedlings in an insectary (27 ± 1°C, 75 ± 5% RH, 16:8 L:D photoperiod) until pupation. Pupae were separated as males and females into 35 x 25 x 25 cm cages. Three-day-old unmated moths were used in all bioassays. Healthy moths were used only once and starved for 12 hr before testing.
Electroantennogram Recordings. For bioassays, the tested chemicals were dissolved individually in normal hexane. The sex pheromone and host-plant volatiles were mixed at ratios of 1:1, 1:10, 1:100, and 1:1000. The concentration of the sex pheromone solution was 50 μl/L for all tests. Solely sex pheromone was used as a control. These solutions were used for EAG as well as for behavior assays. EAG responses of 3-day-old unpaired male/female moth antennae were measured after excised from its base, and the distal part of the terminal segment was cut off. The antenna was xed on the PR electrode with SpectraR360 conductive adhesive at the outlet of the glass tube. In general, 10 μl of the liquid sample was applied to both sides of a lter paper piece. Meanwhile, the lter paper was inserted into a glass tube. An airstream of 0.5-sec duration was blown over the antennae, and EAG responses were recorded. The stimulation interval was 60 s using a ow rate of 1 L/min for both stimulants and purge air ow.
One antenna from each individual was tested with three repeated stimulations. Six antennae were tested for each stimulant. The EAG system used in our experiment is IDAC-232 produced by Syntech (Hilversum, The Netherlands). EAG responses from isolated antennae were recorded according to the method described by Hao (Hao et al. 2006). Data were analyzed using EAG2000 for Windows (SYNTECH, Hilversum, The Netherlands).
Mixtures of the sex pheromone and host-plant volatile at ratios of 1:1, 1:10, 1:100, and 1:1000 were tested. Solely sex pheromone was used as a control. Lures were formulated by applying 10 μl of liquid sample applied to each lter paper before the bioassays. The lure was then pinned to a steel jack for testing. Each lure was used once. 3-day-old moths were transferred individually to test tubes and then introduced into the tunnel individually. The airspeed was 30 cm/sec.
The trap-loaded lures were placed about 20 cm above the plant canopy. The dispenser was a capillary with 90 mg of chemical attractants, which were stored in a freezer before use. The layout of the trial followed a completely randomized block design. The distance between traps was about 25m to minimize interference. Lures were replaced every four days. Insects captured in the traps were removed every two days, counted, sexed, and cataloged. Each treatment was replicated six times in six blocks.
Statistical Analysis. EAG values and trapping data from experiments conducted in the rice paddy were statistically evaluated by one-way ANOVA followed Tukey tests (P<0.05). The responding percentage in wind tunnel experiments was analyzed using a χ2 test.

Result
Effect of Host-Plant Volatiles on EAG Response to Sex Pheromone. Different EAG responses of male or female antennae to the mixtures of sex pheromone and various host-plant volatiles compounds were observed. The EAG values of C. medinalis female moths to the mixtures of sex pheromone and (E)-2-Hexenal increased by 834% and 646% at ratios of 1:100 and 1:1000 compared with sex pheromone alone (F 4,25 = 7.91; P = 0.0003; Table 1), indicating the strongest enhancing effects among the combination of all the tested host-plant volatiles and sex pheromone. Furthermore, the EAG values of C. medinalis female moths increased by 312% and 83% for sex pheromone and (Z)-3-Hexenal mixture at a ratio of 1:1000 (F 4,25 = 9.039; P = 0.0001; Table 1) and for sex pheromone and Valeraldehyde mixture at a ratio of 1:100 (F 4,25 = 3.058; P = 0.0351; Table 1) compared with sex pheromone alone. Signi cantly stronger EAG responses of male moths were observed for sex pheromone and (E)-2-Hexenal mixtures at ratios of 1:100 and 1:1000 (F 4,25 = 5.747; P = 0.02; Table 2) compared with sex pheromone alone. The EAG values increased by 267% and 305%, respectively. The mean response value of males to sex pheromone and Methyl Salicylate mixture at a ratio of 1:100 (F 4,25 = 12.361; P = 0.0001; Table 2) was 97% higher than the response to sex pheromone alone.
Wind Tunnel Bioassay under Laboratory Conditions. Based on the results obtained from EAG tests, (Z)-3-Hexenal, (E)-2-Hexenal, Valeraldehyde, and Methyl Salicylate were selected as candidates for wind tunnel bioassay. For activation and take-off behavior, all mixtures of sex pheromone and (E)-2-Hexenal or Methyl Salicylate (at ratios of 1:1, 1:10, 1:100, and 1:1000) signi cantly increased the response level of females compared with the sex pheromone alone (P < 0.05; Table 4). When mixed with sex pheromone, Valeraldehyde also had a signi cant enhancing effect on the proportion of females activation (at ratios of 1:1 and 1:10) and take off (at ratios of 1:10 and 1:1000) (P < 0.05; Table 4). (E)-2-Hexenal and Methyl Salicylate added to sex pheromone raised the proportion of females partial ight (P < 0.05; Table 4) expect the percentage of 100cm partial ight and 150cm partial ight at a ratio of 1:1000 (P > 0.05; Table 4).
Mixtures of sex pheromone and (Z)-3-Hexenal treatments caused signi cantly fewer males to y upwind and land on the source than the sex pheromone alone (P < 0.05; Table 5). When the mixtures of sex pheromone and Valeraldehyde were used, the response level of all behavioral categories by males was signi cantly decreased at a ratio of 1:100, and the percentage of activation was declined at all concentrations compared with sex pheromone alone (P < 0.05; Table 5). Speci cally, the response level of males activation achieved 100 percent when mixtures of sex pheromone and (E)-2-Hexenal or Methyl Salicylate at ratios of 1:10 and 1:100. At the same time, in the presence of (E)-2-Hexenal (at a ratio of 1:100) and Methyl Salicylate (at ratios of 1:10 and 1:100), the response level of land on by males was signi cantly raised compared with the sex pheromone alone (P < 0.05; Table 5). Across all treatments in wind tunnel assays, only (E)-2-Hexenal mixtures and Methyl Salicylate mixtures increased the attractiveness of both males and females to sex pheromone.
Field Trapping E ciency of Different Compounds. Wind tunnel assays revealed that the mixtures of sex pheromone and (E)-2-Hexenal or Methyl Salicylate increased the proportion of males and females landing on the cage containing the lure compare with sex pheromone alone. Therefore, sex pheromone plus various ratios of these two host-plant volatiles were evaluated in the eld. The data recorded once every two days was recorded 15 times in total. Among them, traps baited with a combination of sex pheromone and Methyl Salicylate at ratios of 1:1 and 1:10 were signi cantly raised male moths catches compare with sex pheromone alone 13 times and 8 times respectively. The quantity of male moths caught by the mixtures of sex pheromone and (E)-2-Hexenal at ratios of 1:1 and 1:10 was signi cantly higher than that of sex pheromone alone 5 times and 13 times respectively (Fig. 1).
In the surveyed eld, traps baited with the mixtures of sex pheromone and (E)-2-Hexenal at ratios of 1:1 and 1:10 signi cantly increased the total number of male moths catch by 94.5% and 109.8%, respectively, compared with traps baited with sex pheromone alone (F 4,15 = 27.079; P = 0.0001; Fig. 2). Male moths caught by the mixtures of sex pheromone and (E)-2-Hexenal between the ratios of 1:1 and 1:10 showed no signi cant difference. Similar to (E)-2-Hexenal, traps baited with the mixtures of sex pheromone and Methyl Salicylate at ratios of 1:1 and 1:10 both captured signi cantly more male moths than the number caught in traps baited with sex pheromone alone (increased by 69% and 146%) (F 4,15 = 37.588; P = 0.0001; Fig. 2). However, the combination of sex pheromone and Methyl Salicylate at a ratio of 1:1 traped fewer male moths than the mixtures of sex pheromone and Methyl Salicylate at a ratio of 1:10. Traps baited with a combination of sex pheromone and (E)-2-Hexenal or Methyl Salicylate at a 1:100 ratio did not raise catches compared with traps baited with sex pheromone alone. Unexpectedly, few female moths are trapped by lures. Moreover, only the lures with (E)-2-Hexenal or Methyl Salicylate almost can not attract C. medinalis moths.

Discussion
Our study demonstrates that (E)-2-Hexenal increased responses of C. medinalis to sex pheromone in EAG, wind tunnel, and eld trapping experiments. (E)-2-hexenal has been reported to enhance the attraction of some other Lepidoptera to their sex pheromones. Deng et al. (2004b) have shown that traps baited with (E)-2-hexenal and sex pheromone enhanced Spodoptera exigua catches by 38.8% compared to traps baited with sex pheromone alone in eld tests. Solely (E)-2-hexenal was not attractive to Leguminivora glycinivorella males at any dose tested but showed a strong synergistic effect when blended with sex pheromone in the eld (Hu et al. 2013). Our data indicated that the ratio between sex pheromone and (E)-2-hexenal could have an impact on the synergistic effect. When blended at ratios of 1:1 and 1:10, the mixtures increased the attraction of C. medinalis male moths to the sex pheromone in eld experiments. It is worthwhile mentioning that in the EAG and wind tunnel experiments, the response concentration is not consistent with that of the eld tests.
This discrepancy could be attributed to different dosages, backgrounds, and bait carriers used. The ratio of host-plant volatiles should deserve attention when combing with sex pheromone.
We observed that Methyl Salicylate also had a synergistic effect on C. medinalis sex pheromone. Deng et al. (2004a) reported that a combination of Methyl Salicylate and sex pheromone elicited stronger EAG responses from Helicoverpa armigera male antennae than sex pheromone alone. Besides, Von Arx et al. (2012) have shown that Methyl Salicylate affected only the initial behavioral responses of Lobesia botrana to sex pheromone in wind tunnel tests. Similarly, low doses (at ratios of 1:1 and 1:10) of Methyl Salicylate enhance the attraction of C. medinalis to sex pheromones, while high doses (at a ratio of 1:100) may not affect the C. medinalis male moths response. Our results and previous studies suggest that (E)-2-hexenal and Methyl Salicylate enhance the response of C. medinalis male moths and some other Lepidoptera to their sex pheromones, and these two compounds may be used as synergists with sex pheromone baits for controlling Lepidoptera pests.
So far, the enhancement of attraction to sex pheromone by host-plant volatiles has been frequently reported in male moths trapping, but there are only a few reports in female moths. Field studies indicate that traps baited with sex pheromone + (Z)-3-hexenyl acetate + (Z)-3-hexen-1-ol + allyl isothiocyanate has a stronger attraction to both males and females Plutella xylostella compare with sex pheromone alone (Dai et al. 2008). A possible explanation for the eld trapping results is those female moths may locate feeding and oviposition sites by responding to host-plant volatiles, and male moths may locate host sites as well as an area of possible female activity by responding to host-plant volatiles and sex pheromone (Bruce et al. 2005). Another view is that male moths use defensive host-plant volatiles as a sex pheromone, which recognition and preference by female moths (Nishida 2014). The combined results of EAG and wind tunnel showed that the mixtures of sex pheromone and (E)-2-hexenal or Methyl Salicylate could increase the response of C. medinalis females to sex pheromone, but almost no females were trapped in the eld. We assumed that it is di cult for female moths to recognize an individual (E)-2-hexenal and Methyl Salicylate in natural conditions due to the high background noise in the eld.
The olfactory system plays an essential role in insect behavior, such as detecting food, mates, oviposition sites, and avoiding enemies (Sato and Touhara 2008). Insects mainly rely on olfaction-related proteins to recognize and percept semiochemicals from the external environment ). The soluble odorant-binding proteins (OBPs) are thought to facilitate the transport of odorant molecules through the sensillum lymph. On the olfactory receptor neurons (ORNs) dendritic membrane, odorant receptors (ORs) linked with the complex of OBP/odors to detect and discriminate distinct odorants (Leal 2013).
Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a uorescent probe demonstrated that trans-11-tetradecen-1-yl acetate, the sex pheromone component of Loxostege sticticalis, and (E)-2-Hexenal, the most abundant plant volatiles in essential oils extracted from host plants, had high binding a nities to LstiGOBP2 (Yin et al. 2012). Grapholita molesta OBP12 (GmolOBP12) displayed a higher expression level in male antennae than in female antennae. Fluorescence binding assays exhibited that GmolOBP12 had strong binding a nities to the sex pheromone (Z)-8-dodecenyl alcohol and host-plant volatile (E)-2-hexenal . Furthermore, it has been reported that Epiphyas postvittana OR1 (EpOR1) is closely related to the pheromone receptors (PR) of other lepidopterans and sensitive to Methyl Salicylate, recognizing this odorant to a low concentration of 10-15M (Zhang et al. 2017). We speculated that the synergism between host-plant volatiles and sex pheromones might be related to the olfactory protein that responds to both odors. It needs further study to con rm our speculation.
In summary, we have demonstrated that the addition of (E)-2-Hexenal or Methyl Salicylate to the sex pheromone bated traps can increase trap catches of C. medinalis male moths in the eld. We believe that apart from looking for the molecular mechanism of the olfactory system, future research should explore food-based synthetic attractant trapping female moths because of their signi cant role in oviposition. These ndings may provide the basis for developing e cient pest management systems against C. medinalis. Note SP sex pheromone, HPV host-plant volatiles; Treatment with different letters in the same line indicates cases in which means for the sex pheromone + host-plant volatiles treatments and sex pheromone alone were signi cantly different (Tukey test, P < 0.05).   Note SP sex pheromone, HPV host-plant volatiles; Data are the percentages of behavioral items carried out within 5 mins; Asterisks among a behavioral response in the same line indicate differences between the blend and individual sex pheromone (P ≤ 0.05*, P ≤ 0.01**, P ≤ 0.001***). Note SP sex pheromone, HPV host-plant volatiles; Data are the percentages of behavioral items carried out within 5 mins; Asterisks among a behavioral response in the same line indicate differences between the blend and individual sex pheromone (P ≤ 0.05*, P ≤ 0.01**, P ≤ 0.001***).    Figure 1 The attraction of C. medinalis male moths to sex pheromone (SP) in combination with the individual (E)-2-Hexenal (a) or Methyl Salicylate (b) in a rice paddy.

Tables
Synthetic sex pheromone combined with (E)-2-Hexenal (a) or Methyl Salicylate (b) at different concentrations (at a ratio of 1:1, 1:10, and 1:100). The mean number of C. medinalis captured in different traps under eld conditions. Treatments were replicated six times in trials. We recorded the number of moths captured by each lure every two days. Asterisks indicate statistically signi cant differences between mixtures and sex pheromone alone (Tukey test; * P < 0.05).

Figure 2
Total number of C. medinalis male moths captured in traps with mixtures of the sex pheromone (SP) in combination with (E)-2-Hexenal (a) or Methyl Salicylate (b) at different concentrations (at a ratio of 1:1, 1:10, and 1:100) in a rice paddy. Signi cant differences between different treatments were analyzed by one-way ANOVA followed by Tukey tests (P < 0.05). Signi cant differences are marked with different letters.