Larval frass in herbivorous lepidopterans is mainly composed of plant-derived material and microbes from the gut. Despite the fact that frass from conspecific larvae repels female moths in Spodoptera littoralis from oviposition, the role of frass volatiles on larval foraging behavior is largely unknown. Here, we show that larvae of S. littoralis walk upwind to larval frass volatiles in a wind tunnel assay. We identified the frass volatile guaiacol (2-methoxyphenol) as key ligand for the S. littoralis odorant receptor (OR) SlitOr59 which we expressed heterologously. We isolated guaiacol-producing bacteria identified as Serratia marcescens from frass of larvae that were fed on cotton, and Enterobacter cloacae, E. ludwigii and Klebsiella sp. from frass derived from cabbage-fed larvae. In addition to guaiacol, we also identified volatiles acetoin, 3-methyl-1-butanol and dimethyl disulfide, in large proportions in headspace collections from the bacteria. A Y-tube olfactometer assay showed that fourth instar S. littoralis larvae are attracted to guaiacol. Moreover, cotton leaves treated with the insecticide Spinosad and guaiacol were highly attractive to the larvae. Our results provide a basis for management of the pest by directly targeting larvae, based on an attract-and-kill strategy. Further studies are needed to test the application of guaiacol for semiochemical-based pest management of Spodoptera pest species.

Background The detection of environmental cues and signals via the sensory system directs behavioral choices in diverse organisms. Insect larvae rely on input from the chemosensory system, mainly olfaction, for locating food sources. In several lepidopteran species, foraging behavior and food preferences change across larval instars; however, the molecular mechanisms underlying such behavioral plasticity during larval development are not fully understood. Here, we hypothesize that expression patterns of odorant receptors (ORs) change during development, as a possible mechanism influencing instar-specific olfactory-guided behavior and food preferences. Results We investigated the expression patterns of ORs in larvae of the cotton leafworm Spodoptera littoralis between the first and fourth instar and revealed that some of the ORs show instar-specific expression. We functionally characterized one OR expressed in the first instar, SlitOR40, as responding to the plant volatile, β-caryophyllene and its isomer α-humulene. In agreement with the proposed hypothesis, we showed that first but not fourth instar larvae responded behaviorally to β-caryophyllene and α-humulene. Moreover, knocking out this odorant receptor via CRISPR-Cas9, we confirmed that instar-specific responses towards its cognate ligands rely on the expression of SlitOR40. Conclusion Our results provide evidence that larvae of S. littoralis change their peripheral olfactory system during development. Furthermore, our data demonstrate an unprecedented instar-specific behavioral plasticity mediated by an OR, and knocking out this OR disrupts larval behavioral plasticity. The ecological relevance of such behavioral plasticity for S. littoralis remains to be elucidated, but our results demonstrate an olfactory mechanism underlying this plasticity in foraging behavior during larval development.