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.