TABLES
Table 1. Best BLAST hits for candidate CXEs identified in the chemosensory organs of C. molesta .
Table 2. Motif analysis and biochemical characteristics of the 15 putativeGmolCXEs with full-length sequences.
E: Glutamate; H: Histidine; SP: signal peptide; MW: molecular weight; PI: isoelectric point; Y: Yes; N: No.
FIGURES
Figure 1. Phylogenetic tree analysis of candidate GmolCXEs in the C. molesta. The tree was constructed with MEGA 8.0 using the neighbor-joining method. The values at the nodes are the results of bootstrapping with 1,000 replicates. GmolCXE genes are shown in red. Different color regions represent different categories of CXEs in phylogenetic tree. All the candidate GmolCXEs distributed into the designate clades of phylogenetic tree according to the classification system in reported references (Oakeshott et al., 2005; Durand et al., 2010).The accession numbers in GenBank and protein names of other species CXEs involved in tree are listed in Table S1.
Figure 2. qRT-qPCR analysis of GmolCXEs in male/female antennae or male antennae after female moth stimulation. A: Relative mRNA levels of 23 GmolCXE s in male and female antennae. B and C: The spatial expression patterns ofGmolCXE1 (B) and GmolCXE5 (C) in different tissues of male and female adults. D: Relative expression analysis of 23 GmolCXE s in male antennae after female adults stimulation.
Figure 3. qRT-PCR analysis of GmolCXEs in larva head/ abdomen or larva head after fresh fruit stimulation. A: Relative mRNA expressions of 23 GmolCXE s in the head and abdomen of 4th instar larvae. B: Relative mRNA expressions of 23GmolCXE s in the 4th instar larvae after fresh fuji apples and crown pears stimulation by qRT-PCR.
Figure 4. Behavioral assays after GmolCXE1 andGmolCXE5 of male adults knocked down by RNAi. A and B: The relative expression within 72 hours of GmolCXE1 (A) andGmolCXE5 (B) in male antenna after injected with the corresponding dsRNAs. C: Comparison of the numbers of individuals that find female adults. D: Time spent finding female moths byGmolCXE1 and GmolCXE5 silencing respectively. E: Frequency of male moths turning back in Y-tube searching for female adults. F: EAG response to sex pheromone mix components of female moths. “n” showed the number of larvae that did not respond to female moths or water.
Figure 5. Electrophysiological assays analysis for esterase treated sex pheromone components. A, B and C: Male moths’ EAG responses to two sex pheromone components and mix components treated with purified GmolCXE1 and GmolCXE5 respectively. Z8-12:Ac:(Z )-8-dodecenyl acetate. E8-12:Ac:(E )-8-dodecenyl acetate. Mix: Z8-12:Ac: E8-12:Ac=9:1.
Figure 6. Degradation analysis of GmolCXE1 and GmolCXE5 with sex pheromone components by GC-MS. A and E: Control group. B and F: The two ester sex pheromone components treated with purified GmolCXE1. C and G: The two ester sex pheromone components treated with purified GmolCXE5. D and H: Percent of degradation on two ester sex pheromone components treated with purified GmolCXE1 and GmolCXE5 respectively. RT: retention time. PA: peak area. Percent of degradation (%) = (1- peak area of alcohol/ peak area of control)*100
Figure 7. Behavioral assays after GmolCXE13,GmolCXE14 and GmolCXE21 of larvae knocked down by RNAi.A, B and C: The relative expression within 72 hours of GmolCXE13 ,GmolCXE21 and GmolCXE14 after larvae injected with the corresponding dsRNA. D and F: Comparison of the numbers of individuals that find fresh fruits after GmolCXE13 , GmolCXE21 andGmolCXE14 silencing respectively. G and I: Time spent searching for food after GmolCXE13, GmolCXE14 and GmolCXE21silencing respective. “n” showed the number of larvae that did not respond to food or water.
Figure 8. Behavioral assays of larvae responses to two ester volatiles from ripe crown pears and degradation analysis of GmolCXE14 and GmolCXE21 with two ester volatiles by GC-MS. A: Behavioral assays of larvae responses to ethyl butanoate and ethyl hexanoate by Y-tube. B and C: Behavioral assays of larvae responses to ethyl butanoate and ethyl hexanoate after GmolCXE14 and GmolCXE21 knocked down respectively. D and G: Control group. E and H: The two ester sex pheromone components treated with purified GmolCXE14. F and I: The two ester sex pheromone components treated with purified GmolCXE21. J and K: Percent of degradation on two ester volatiles treated with purified GmolCXE14 and GmolCXE21 respectively. RT: retention time. PA: peak area. Percent of degradation (%) = (peak area of ester volatiles-treatment / peak area of ester volatiles-control)*100
Figure S1. Diagrammatic drawing of fourth instar larvae or male adults by odor-stimulation. A: The fourth instar larvae were stimulated by ripe fuji apples or crown pears. B: The male moths were stimulated by female moths.
Figure S2. The protein sequences analysis of 15 full-lengthGmolCXEs by ClustalX 2.0. The shaded labeled region in the figure were the conserved motif of 15 full-length GmolCXEs. The conserved motif-pattern with the motif order Oxyanion-Ser active site-Glu-His.
Figure S3. Expression and purification of GmolCXEs were testedusing SDS-PAGE analysis. A, B, C and D: Expression and purification of GmolCXE1, GmolCXE5, GmolCXE14 and GmolCXE21 respectively. M: TransBlue Plus TM Protein Marker. 1: Expression of proteins without IPTG. 2: Expression in the supernatant of proteins with 1.0 mM IPTG after ultrasonic crushing. 3: Expression in the inclusion body of proteins with 1.0 mM IPTG after ultrasonic crushing. 4: Purified proteins with His-tag.
Figure S4. Chromatogram and mass spectrogram of two eater volatiles from ripe crown pears were analyzed by GC-MS. A: Collection time analysis of odor volatiles from ripe crown pears. B and C: Chromatogram and mass spectrogram of ethyl butanoate respectively. D and E: Chromatogram and mass spectrogram of ethyl hexanoate respectively.