Yeast-derived volatiles orchestrate an insect-yeast mutualism with oriental armyworm moths

Abstract

Interactions among insects, plants, and microorganisms are fundamental to ecosystem dynamics, with floral nectar and pollen serving as key resources for various organisms. Yeasts, such as Metschnikowia reukaufii, commonly found in nectar, influence nectarial attraction through volatile compounds (VOCs), yet the underlying biological mechanisms remain elusive. Here, we show that isoamyl alcohol, a prominent yeast VOC, attracts oriental armyworm moths (Mythimna separata) to pollen-rich, yeast-fermented nectar. In a series of electrophysiological and behavioral assays, we show that isoamyl alcohol activates a single class of highly specific olfactory sensory neurons expressing the olfactory receptor MsepOR8. In the moth antennal lobe, these neurons target the AM2 glomerulus, which responds to isoamyl alcohol. Genetic disruption of MsepOR8 leads to complete abolition of both physiological and behavioral responses to isoamyl alcohol, resulting in an impaired ability to locate nectar sources. Moreover, we show that isoamyl alcohol-induced foraging behavior fosters a mutualistic relationship between yeast and moths to some extent, enhancing yeast dispersal and increasing moth reproductive success. Our results unveil a highly specific mechanism by which a yeast-derived VOC facilitates insect-yeast mutualism, providing insights into insect-microbe interactions within pollination ecosystems.

Fig. 1. Attraction of oriental armyworm moths to pollen-rich, yeast-fermented nectar.

a Schematic drawing of the wind tunnel. b Female oriental armyworm moth behavioral responses to various stimuli: water (H₂O), pure nectar (NEC), yeast-fermented nectar (NEC + Y), pollen-rich nectar (NEC+Rape P, NEC+Sunflower P, NEC+Maize P), and pollen-rich yeast-fermented nectar (NEC + Y+Rape P, NEC + Y+Sunflower P, NEC + Y+Maize P). n = 5 replicates, each replicate consisted of 5–8 individuals. p = 2.825 × 10⁻¹¹ in close search and p = 3.369 × 10⁻¹¹ in landing, one-way ANOVA followed by Tukey’s test. Error bars represent the standard error of the mean (SEM). c Male oriental armyworm moth behavioral responses to the same stimuli as described in (b). n = 5 replicates, each replicate consisted of 5–7 individuals. p = 8.776 × 10⁻⁹ in close search and p = 4.294 × 10⁻¹⁰ in landing, statistical analysis and error bars were performed as mentioned. Source data are provided as a Source Data file.

Fig. 2. Isoamyl alcohol is the principal attractant for oriental armyworm moth in pollen-rich, yeast-fermented nectar.

a Schematic drawing of odor collection by solid-phase microextraction (SPME) for gas chromatography–mass spectrometry (GC–MS) and gas chromatography-electroantennographic detection (GC-EAD). b Gas chromatograms showing the composition of volatiles emanating from different nectar sources: pure nectar (NEC), yeast-fermented nectar (NEC + Y), pollen-rich nectar (NEC+Rape P, NEC+Sunflower P, NEC+Maize P), and pollen-rich yeast-fermented nectar (NEC + Y+Rape P, NEC + Y+Sunflower P, NEC + Y+Maize P). c Electroantennographic responses of female and male oriental armyworm moth antennae to different pollen-rich, yeast-fermented nectars, including NEC + Y+Rape P, NEC + Y+Sunflower P, NEC + Y+Maize P, linked with gas chromatography. Flame Ionization Detector (FID). d Electroantennogram responses to isoamyl alcohol and volatile organic compounds (VOCs) from NEC + Y+Rape P. n = 25 antennae (one antenna was used from each moth). p = 1.121 × 10⁻²⁶ in females and p = 8.683 × 10⁻³⁷ in males, one-way ANOVA followed by Tukey’s test. Error bars represent the standard error of the mean (SEM). e Behavioral responses of oriental armyworm moths to different stimulus in a wind tunnel including H₂O, NEC, isoamyl alcohol and VOCs from NEC + Y+Rape P. n = 5 replicates, each replicate consisted of 5–8 individuals, p = 4.537 × 10⁻⁷ in close search and p = 1.713 × 10⁻⁷ in landing in females, p = 4.0 × 10⁻⁶ in close search and p = 6.688 × 10⁻⁷ in landing in males, statistical analysis and error bars were performed as mentioned. In all experiments, EtOH (ethanol) and isoamyl alcohol were used at concentrations consistent with those found in NEC + Y+Rape P, with 1.1% (v/v) and 0.00044% (v/v), respectively. Numbers at the peaks in panels b and c denote (1) Ethanol (EtOH); (2) 2-methyl-1-propanol; (3) isoamyl alcohol. Source data are provided as a Source Data file.

Fig. 3. Isoamyl alcohol activates a single type of olfactory sensory neuron expressing MsepOR8.

a Schematic drawing of single-sensillum recording and sequencing. b Single-sensillum recordings showing three neurons housed in sensilla auricillica, with the 3rd neuron (characterized by the largest spikes) responding to isoamyl alcohol. c Single-sensillum sequencing reveals the expression of four MsepORs plus Orco in cells under auricillica sensilla. d Electrophysiological responses of oocytes expressing MsepOR8/MsepOrco and MsepOR27/MsepOrco to a 10⁻⁴ M concentration of isoamyl alcohol revealing MsepOR8 as the isoamyl alcohol-detecting receptor. e Electrophysiological responses of oocytes expressing MsepOR8/MsepOrco to 32 odorants at a 10⁻⁴ M concentration showing the high specificity of the interaction between MsepOR8 and isoamyl alcohol. f Dose response of MsepOR8/MsepOrco-expressing oocytes to isoamyl alcohol. EC50 = 1.74 × 10⁻⁶ M. g Calcium imaging recordings from the antennal lobe showing specific isoamyl alcohol-induced calcium responses in the AM2 glomerulus. The white and black figure is the transmission image of the same field, and the white dashed line in the image represents the recorded field of the antennal lobe. n = 4. h Neural tracing reveals three innervated glomeruli out of which one is the AM2 glomerulus. n = 3. In d–f, replicate numbers are indicated (n = 9 for (d), n = 7–20 for (e), n = 3–6 for (f)), and error bars represent the standard error of the mean (SEM). Source data are provided as a Source Data file.

Fig. 4. MsepOR8 is essential for the response of oriental armyworm moth to isoamyl alcohol.

a Electroantennographic (EAG) response comparison between wild-type (WT) and MsepOR8 knockout (MsepOR8^−/−) oriental armyworm moths to isoamyl alcohol. n = 25, p = 0.684 in NEC (pure nectar) stimuli and p = 2.662 × 10⁻¹¹ in isoamyl alcohol stimuli. b Single-sensillum recording (SSR) responses from sensilla auricillica in WT and MsepOR8^−/− oriental armyworm moths to NEC + Y+Rape P (rape pollen-rich yeast-fermented nectar) and isoamyl alcohol. n = 12, p = 0.225 in NEC stimuli, p = 1.292 × 10⁻¹⁰ in NEC + Y+Rape P stimuli and p = 2.040 × 10⁻¹⁰ in isoamyl alcohol stimuli. c Calcium responses among antennal lobe glomeruli of WT and MsepOR8^−/− oriental armyworm moths to NEC + Y+Rape P and isoamyl alcohol. The white and black figure is the transmission image of the same field, and the white dashed line in the image represents the recorded field of the antennal lobe. n = 3. d Behavioral response comparison between WT and MsepOR8^−/− oriental armyworm moths to NEC + Y+Rape P and isoamyl alcohol. n = 5 replicates, each containing 5–8 individuals, in close search, p = 6.0 × 10⁻⁶ in NEC + Y+Rape P stimuli and p = 1.2 × 10⁻⁵ in isoamyl alcohol stimuli, in landing, p = 9.0 × 10⁻⁵ in NEC + Y+Rape P stimuli and p = 2.1 × 10⁻⁵ in isoamyl alcohol stimuli. Statistical analyses in panels a, b, and d were performed using two-sided t-tests. Error bars represent the standard error of the mean (SEM). Source data are provided as a Source Data file.

Fig. 5. Isoamyl alcohol-mediated foraging behavior promotes the dispersal of yeast and augments the reproduction of oriental armyworm moth.

a Female MsepOR8^−/− oriental armyworm moth exhibit a significantly reduced visiting rate to rape pollen-rich yeast-fermented nectar (NEC + Y+Rape P) compared to wild-type individuals. n = 35 replicates in both wild-type group and MsepOR8^−/− group, p = 9.76 × 10⁻⁴, statistical analyses were performed using a two-sided t-test. Error bars represent the standard error of the mean (SEM). b Female MsepOR8^−/− oriental armyworm moth exhibit significantly reduced capacity to disseminate yeasts from NEC + Y+Rape P compared to wild-type individuals. n = 33 replicates in the wild-type group and n = 31 replicates in the MsepOR8^−/− group, p = 0.0025 and analyzed using the Chi-square test in the above column chart, p = 0.00105 and analyzed using the two-sided t-test in the below column chart. Error bars represent the standard error of the mean (SEM). c Pollen significantly augments the fecundity of female oriental armyworm moth. n = 4 replicates per treatment, and each replicate comprised a cohort of newly emerged moths consisting of 10 females and 12 males. p = 2.8 × 10⁻⁵, one-way ANOVA followed by Tukey’s test. Error bars represent the standard error of the mean (SEM). Source data are provided as a Source Data file.