# Combinatorial Olfactory Signaling in Short-Distance Determines Host Plant Recognition in Locust
## 4 Discussion  
How insect herbivores select the correct plants to eat is an unresolved and crucial question in insect-plant interactions. Several hypotheses and theories have been proposed  
Figure 4: Multiple odorant receptors (ORs) are required for plant odors discrimination. (**A**): PI_DER to wheat grinds in wild-type locusts (WT) or locusts injected with water (ddH\({}_{2}\)O) or dsRNA of GFP (dsGFP). \(N=10\)–\(18\). (**B**): PI_DER to wheat grinds in locusts injected with dsRNA against multiple ORs. \(N=10\)–\(22\). (**C**-**F**): PI_DER to single odorants in locusts injected with dsRNA against OR12 (**C**). OR22 (**D**), OR15 (**E**), and OR21 (**F**). Chemical dose: 15% (\(\
u/\
u\)). \(N=10\)–\(23\). (**G**): Working model of combinatorial olfactory signaling of host-plant recognition by a locust based on the present results. One-way ANOVA with Fisher’s LSD multiple comparisons tests. Error bars represent the SEM. \({}^{*}p<0.05\), \({}^{**}p<0.01\), \({}^{***}p<0.001\), \({}^{***}p<0.0001\). to account for this common ecological communication process in different insect-plant combinations [1; 14; 40; 41; 42]. However, how the selection strategy was shaped and diverged among insect groups remains enigmatic. This study revealed that short-range olfactory coding to characteristic volatiles in both host and nonhost plants facilitated critical decision-making with regard to food sources in the oligophagous locust. An olfactometer is widely used to evaluate the behavioral effects of plants on insect feeding preference, for instance, Y-tube or T-tube types [43; 44]. The insects are observed for their response to loaded volatiles in an open arena, and several parameters are measured, such as the preference to approach each loaded volatile and the time or distance they stay in corresponding spaces. One of the drawbacks of these traditional paradigms is the uncertainty of where and when the olfactory process occurs, which is of particular importance to pinpoint the insect-plant interaction strategy. The DER is designed to visualize the olfactory contribution of locust palps to food cues at short range. Locusts move freely in the tunnel offering two choices containing volatiles, and then vibrate the palps intensely in close proximity to an attractive stimulus, which is reminiscent of palpation. Using DER in this study, we observed that oligophagous locusts sensitively discriminated hedonic wheat extraction from repulsive cotton extraction, and this selection depended on short-range olfactory pathways on the palps. The DER assay allowed us to release the restricted insects and observe their olfactory choice toward stimulus delivery at a short range. This assay could also be modified for testing other insect herbivores. It has long been considered that volatile cues emitted by plants contribute largely to influencing insect behavior during host plant searching and selection [13; 15; 21; 45]. Herbivorous insects are exposed to plant volatiles in a complex environment, but not all plant volatiles may carry information that is useful for host-plant decisions. With consideration of polyphago insects, in particular, there can be few or even no common volatiles that are indicative of the plant spectrum of interest in a broad range of host plants. Nevertheless, oligophagous insects are thought to choose food plants relatively more easily because limited hosts are associated with specific attractive infochemicals that are indicative of plant localization. It has been reported that insects appear to choose host plants via recognition of specific blends of certain ubiquitous odors. The signal value of this blend seems to be interpreted by the blend composition as a whole instead of the summed effects of individual components [42]. Although greater focus has been paid to the identification and behavioral tests of host-plant-derived volatiles [46; 47], whether nonhost plants release inhibitory odors to repel oligophagous insects remains unclear.