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BMC Biology
Expression ofapheromone binding protein
aected bytimeless gene governs female
mating behavior inBactrocera dorsalis
Yuting Jiao1†, Guohong Luo1†, Yongyue Lu1 and Daifeng Cheng1*
Abstract
Background The rhythmic mating behavior of insects has been extensively documented, yet the regulation of this
behavior through sex pheromone sensing olfactory genes affected by the clock genes in the rhythm pathway
remains unclear.
Results In this study, we investigated the impact of circadian rhythm on female recognition of male rectal Bacillus-
produced sex pheromone in B. dorsolis. Behavioral and electrophysiological assays revealed a peak in both mating
behavior and response to sex pheromones in the evening in females. Comparative transcriptome analysis of female
heads demonstrated rhythmic expression of the Timeless gene-Tim and odorant binding protein gene-Pbp5,
with the highest expression levels occurring in the evening. Protein structural modeling, tissue expression patterns,
RNAi treatment, and physiological/behavioral studies supported Pbp5 as a sex pheromone binding protein whose
expression is affected by Tim. Furthermore, manipulation of the female circadian rhythm resulted in increased morn-
ing mating activity, accompanied by consistent peak expression of Tim and Pbp5 during this time period. These
findings provide evidence that insect mating behavior can be modulated by clock genes through their effects on sex
pheromone sensing processes.
Conclusions Our results also contribute to a better understanding of the molecular mechanisms underlying rhyth-
mic insect mating behavior.
Keywords Bactrocera dorsalis, Sex pheromone, Pheromone binding protein, Timeless gene
Background
Reproductive behavior, including courtship, mating,
and oviposition, is a fundamental biological activity for
insects to regulate their populations [1]. Mating behav-
ior serves as the pivotal step in reproductive activity [2]
and is typically initiated following successful courtship
[3]. During courtship, sexually mature individuals utilize
pheromones, visual signals, or vocalizations to attract
potential mates from a distance [4–6]. At close range,
they employ visual, olfactory, and tactile cues to express
affection and facilitate acceptance of mating behavior
from the opposite sex [7].
Environmental factors exert a significant influence on
the mating behavior of insects [8]. e periodic fluctua-
tions in light serve as crucial signaling cues that impact
the courtship and mating behavior of most insects [7,
9]. Environmental changes in light, coupled with tem-
perature variations, represent important entrainment
factors affecting insect mating behavior [10]. e circa-
dian rhythm is an endogenous biological process driven
by molecular oscillations of the internal clock [11].
†Yuting Jiao and Guohong Luo contributed equally to the study.
*Correspondence:
Daifeng Cheng
chengdaifeng@scau.edu.cn
1 Department of Entomology, South China Agricultural University,
Guangzhou 510640, China
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Jiaoetal. BMC Biology (2025) 23:56
Most central clocks undergo endogenous cyclic oscil-
lations within the central nervous system of organisms
[12–14]. Peripheral clocks, under the influence of the
central clock, modulate various behaviors in organisms
[15, 16]. Extensive research has been conducted on the
role of the peripheral nervous system in animal phero-
mone-mediated courtship behaviors [17–19]. Circadian
rhythms governing mating behavior play a pivotal role
in population survival and propagation among animals
[20]. Mating behavior that occurs at a specific time ena-
bles animals to evade adverse environmental conditions
such as predators and concentrate reproductive activi-
ties at opportune times based on their ecological niches,
thereby enhancing safe reproduction and promoting
stable population growth [21]. A comprehensive under-
standing of how and why animals mate at specific times
provides valuable insights into their reproductive strate-
gies, evolutionary adaptations, and ecological interac-
tions, offering guidance for conservation efforts, wildlife
management strategies, and agricultural pest control
initiatives.
Previous research has suggested that male B. dorsalis
release rectal Bacillus produced pyrazine compounds
(trimethylpyrazine (TMP) and tetramethylpyrazine
(TTMP)) as sex pheromones in the evening to attract
females for mating [22, 23]. However, it remains unclear
how females perceive the pheromone signal and whether
their response is limited to the evening. In this study, we
further demonstrate that female mating behavior exhibits
rhythmicity, occurring exclusively in the evening. Elec-
troantennogram (EAG) recordings and behavioral assays
indicate a rhythmic variation in female sensitivity to sex
pheromones, peaking in the evening. Comparative head
transcriptome analysis reveals that the sex pheromone
binding protein-Pbp5 is responsible for binding the sex
pheromone. Furthermore, we show that the clock gene-
Timeless (Tim) can regulate the rhythmic expression of
Pbp5. is study contributes to our understanding of
insect reproductive behavior and serves as an important
supplement to research on insect olfactory perception.
Results
Mating behavior offemales andtheir attraction tosex
pheromone peaking intheevening
To investigate whether female mating occurs at spe-
cific times of the day, we monitored mating frequency
throughout the day. e results revealed that females
only mate in the evening, particularly at 20:00 (Fig.1a).
Previous research has demonstrated that TMP and
TTMP are male-derived sex pheromone that effectively
attracts females [22–24]. We hypothesized that dif-
ferences in female sensitivity to TMP and TTMP may
account for variations in mating frequency at different
times of day. To test this hypothesis, we assessed the
attractiveness of TMP and TTMP to females at various
time points throughout the day. Our findings indicated
that TMP and TTMP strongly attracted females only in
the evening, especially at 20:00 (Fig.1b), consistent with
the observation on mating frequency. Furthermore, EAG
recordings showed that both TMP and TTMP elicited
stronger responses from female antennae in the evening
compared to the morning (Fig.1c–h). ese results sug-
gest that both mating behavior and response to sex pher-
omone peak in the evening.
Screening forgenes associated withrhythmic mating
behavior
To further screen and identify the olfactory and clock
genes that regulate the rhythmic mating of females, we
conducted female head RNA-seq analysis at 4-h inter-
vals throughout the day. e results revealed no sig-
nificant differences in head gene expression patterns
at different times of the day (Additional file1: Fig. S1a,
Additional file2: Dataset S1). However, the comparison
of female head samples collected at different times led
to a greater number of identified differentially expressed
genes (DEGs) (Fig.2a, Additional file1: Fig. S1b–1f and
Additional file 3–7: Datasets S2–S6). Subsequently,
KEGG analysis was performed on most DEGs screened
between 8:00 and 20:00 to identify genes responsible
for regulating rhythmic mating behavior in females. e
results revealed a significant enrichment of the circadian
rhythm pathway (Fig. 2b). Analysis of gene expression
patterns for differentially expressed genes (DEGs) in the
circadian rhythm pathway indicated high expression of
Tim in the heads of females at 20:00 (Fig.2c and e, Addi-
tional file8: Dataset S7). Given that olfactory genes, such
as chemosensory proteins (Csps), odorant binding pro-
teins (Obps), and odorant receptors (Ors), are primarily
responsible for binding volatiles, including sex phero-
mones [25], we analyzed the expression patterns of olfac-
tory genes in female heads at different times to identify
potential genes involved in sex pheromone binding. e
results showed differential expression of various olfactory
genes between heads at 8:00 and 20:00 (Fig. 2d, Addi-
tional file9: Dataset S8). Among these genes, pheromone
binding protein 5 (Pbp5) exhibited the highest and low-
est expressions at 20:00 and 8:00, respectively, suggesting
its role in sex pheromone binding. To further investigate
Pbp5 function, odorant binding protein 99a (Obp99a),
which displayed high expression across all sampling
times, was selected as the control gene for subsequent
functional analysis. qPCR results also demonstrated high
expression of Pbp5 in female antennae at 20:00 (Fig.2f).
ese findings suggest that peak mating behavior among
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Jiaoetal. BMC Biology (2025) 23:56
females at 20:00 may be associated with elevated levels of
Tim and Pbp5.
Pbp5 exhibits robust binding capabilities tobothTMP
andTTMP
e interaction between Pbp5 and the ligands (N-phe-
nyl-1-naphthylamine (1-NPN), TMP, and TTMP) was
predicted using Alphafold 2.0 for homology modeling
of Pbp5, followed by prediction of the interaction sites
with Autodock Vina (Additional file1: Fig. S2a). e
evaluation results indicated a high-quality score of 96.7
for the constructed Pbp5 model (Additional file1: Fig.
S2b), and all amino acids fell within the optimal and
suboptimal regions in the Ramachandran Plot (Addi-
tional file1: Fig. S2c), suggesting suitability for docking
analysis. e docking results revealed that Phe-31, Ile-
52, Lys-55, Phe-95, and Phe-147 constituted the bind-
ing site of Pbp5 to 1-NPN (Fig.3a, Additional file10:
TableS1). Additionally, Ile-28, Phe-31, Phe-95, and Phe-
147 were identified as the binding sites of Pbp5 to TMP
and TTMP (Fig.3b and c, Additional file10: TableS1).
Furthermore, it was found that Phe-31, Phe-95, and
Phe-147 were common binding sites for TMP, TTMP,
and 1-NPN to Pbp5 (Additional file10: TableS1), sug-
gesting that competitive fluorescence binding experi-
ments between TMP (TTMP) and 1-NPN could
be conducted to assess the binding affinity of TMP
(TTMP) to Pbp5. Subsequent ligand binding assays
demonstrated strong interaction between 1-NPN with
recombinant Pbp5 protein with a dissociation constant
(Kd) of 10.57 μM (Fig.3d). Moreover, competitive fluo-
rescence binding experiments indicated that both TMP
and TTMP exhibited stronger affinities towards Pbp5
compared to 1-NPN with the inhibition constant (Ki)
of 0.2243 μM and 0.2101 μM, respectively (Fig.3e and
f). en the recombinant protein of Pbp5 with the pre-
dicted binding sites to TMP and TTMP being mutated
(Ile-28, Phe-31, Phe-95, and Phe-147 mutated to Ala)
were expressed and purified. Subsequent ligand bind-
ing assays showed 1-NPN still had strong binding abil-
ity to the mutant Pbp5 protein with a Kd value of 33.89
μM (Fig.3g). However, competitive fluorescence bind-
ing experiments indicated that both TMP and TTMP
exhibited almost no affinities towards mutant Pbp5
protein with Ki values of 3e + 31 μM and 9e + 24 μM,
respectively (Fig.3h and i). ese findings suggest that
Fig. 1 Female mating behavior and attraction to sex pheromone peak in the evening. a Number of mated females at different time periods in a day
(replicates = 5, F(2,12) = 690.6, P < 0.0001, one-way ANOVA). b Number of females attracted by sex pheromone at different time periods in a day
(replicates = 8, F(2,21) = 54.83, P < 0.0001, one-way ANOVA). c Female EAG response to TMP at 8:00 and 20:00 (replicates = 5, P = 0.0295, independent
sample t test). d Female EAG response to TTMP at 8:00 and 20:00 (replicates = 5, P = 0.0259, independent sample t test). e Female EAG response
to TMP and TTMP mixture at 8:00 and 20:00 (replicates = 11, P = 0.0004, independent sample t test). f Example traces of female antenna responses
to TMP. g Example traces of female antenna responses to TTMP. h Example traces of female antenna responses to mixture of TMP and TTMP
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Jiaoetal. BMC Biology (2025) 23:56
TMP and TTMP can effectively bind to Pbp5 and may
regulate the rhythmic mating behavior of females.
RNAi‑mediated knockdown ofPbp5 results inimpaired
female mating andreduced EAG response toTMP
andTTMP
We then conducted an investigation into the role of
Pbp5 through sequence similarity analysis and RNAi. A
maximum likelihood phylogenetic analysis using amino
acid sequence alignments for 10 Obps from various Dip-
tera species revealed that Pbp5 was highly conserved
within Tephritidae (Fig.4a). e majority of amino acids
were conserved within the selected species, particularly
the four signature cysteine residues of Obps (Fig. 4b).
Gene expression analysis demonstrated that Pbp5 exhib-
ited the highest expression in female antennae, while
Fig. 2 Tim and Pbp5 screened by transcriptome analysis may be associated with peaking mating behavior of females at 20:00 in a day. a The
number of DEGs in the female head at 20:00 compared with those at other times. Up: significantly up expressed genes in the head of females
at 20:00; down: significantly down expressed genes in the head of females at 20:00. b KEGG pathways enriched with the DEGs between female
heads at 20:00 and 8:00. c Expression patterns of DEGs (between 20:00 and 8:00) in the circadian rhythm pathway at different times in a day.
Before heat mapping, the FPKM values of the DEGs were processed using Z-score standardization. (d) Expression patterns of the olfactory genes
in the head of females at different times in a day. Before heat mapping, the FPKM values were processed using Z-score standardization. e Relative
expression verification of Tim in the head of females at different times in a day (replicates = 5, F(5,24) = 3.906, P = 0.0099, one-way ANOVA). f Relative
expression verification of Pbp5 in the head of females at different times in a day (replicates = 5, F(2,24) = 5.866, P = 0.0011, one-way ANOVA)
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Jiaoetal. BMC Biology (2025) 23:56
Obp99a peaked in the female head without antennae
(Fig.4c and d). Subsequent RNAi experiments resulted
in a decrease of more than 50% in the expression levels
of Pbp5 and Obp99a (Additional file1: Fig. S3a and 3b),
with no significant impact on survival observed (Addi-
tional file1: Fig. S3c and 3d). Mating competition assays
revealed that knocking down Pbp5 significantly reduced
female mating number, whereas knocking down Obp99a
had no effect on mating (Fig. 4e and f). Additionally,
EAG responses to TMP, TTMP, and mixture of TMP
and TTMP were significantly reduced in females with
knocked down Pbp5 expression (Fig. 4g–i, Additional
file1: Fig. S4a–4c). ese findings suggest that Pbp5 plays
a role in female mating and response to sex pheromone-
TMP and TTMP.
Tim aects theexpression ofPbp5
Given that both Tim and Pbp5 are rhythmically
expressed, we infer that Tim may influence expression
of Pbp5. To test this hypothesis, we initially examined
the tissue expression of Tim in female flies. e results
revealed that Tim exhibited the highest expression in
the antenna, similar to Pbp5 (Fig. 5a). Knocking down
Tim through RNAi led to a decrease in Pbp5 expression
Fig. 3 Binding ability of Pbp5 to TMP and TTMP. a Binding sites prediction between Pbp5 and 1-NPN by Autodock Vina. b Binding sites prediction
between Pbp5 and TMP by Autodock Vina. c Binding sites prediction between Pbp5 and TTMP by Autodock Vina. d Binding abilities of 1-NPN
to Pbp5. e Competitive binding ability of TMP to 1-NPN and Pbp5 complex. f Competitive binding ability of T TMP to 1-NPN and Pbp5 complex. g
Binding abilities of 1-NPN to Pbp5 mutant. h Competitive binding ability of TMP to 1-NPN and Pbp5 mutant complex. i Competitive binding ability
of TTMP to 1-NPN and Pbp5 mutant complex
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Jiaoetal. BMC Biology (2025) 23:56
(Fig.5b and c). Additionally, knockdown of Tim signifi-
cantly reduced female mating frequency (Fig. 5d) and
EAG response to sex pheromone (Additional file1: Fig.
S5a–5c). To further confirm the relationship between
Tim and Pbp5, we manipulated the circadian rhythm
of female flies by subjecting them to darkness during
daytime and light during nighttime. Subsequent gene
expression analysis demonstrated a complete reversal
in the patterns of Tim and Pbp5 expressions compared
to normal reared flies (Fig. 2e and f), with both genes
exhibiting peak expressions at 8:00 (Fig.5e). Mating com-
petition assays also indicated that fewer females with
Fig. 4 RNAi of Pbp5 reduces female mating and EAG response to TMP and TTMP. a Maximum likelihood topology tree shows Pbp5 is conserved
in Tephritidae. b Conservation of Pbp5 amino acids in Tephritidae. The image was generated using the complete amino acid sequences
of the species in Tephritidae in a. c Female tissue expression of Pbp5 (replicates = 5, F(3,16) = 82.79, P < 0.0001, one-way ANOVA). d Female
tissue expression of Obp99a (replicates = 5, F(3,16) = 263.5, P < 0.0001, one-way ANOVA). e Influence of Obp99a knockdown on female mating
(replicates = 6, P = 0.2722, paired sample t test). f Influence of Pbp5 knockdown on female mating (replicates = 6, P = 0.0047, paired sample t test).
g Influence of Pbp5 knockdown on female EAG response to TMP (replicates = 19, P < 0.0001, independent sample t test). h Influence of Pbp5
knockdown on female EAG response to TTMP (replicates = 19, P = 0.0015, independent sample t test). i Influence of Pbp5 knockdown on female EAG
response to TMP and TTMP mixture (replicates = 19, P = 0.0002, independent sample t test)
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Jiaoetal. BMC Biology (2025) 23:56
reversed circadian rhythms mated with males than con-
trol females in the evening (20:00) (Fig.5f), while more
females with reversed circadian rhythms mated with
males in the morning (8:00) (Fig.5g). Consistently, EAG
responses to sex pheromone decreased significantly for
females with reversed circadian rhythms in the evening
but increased significantly in the morning (Additional
file 1: Fig. S5d–5i). ese findings collectively suggest
that Tim can affect both Pbp5 expression and female
mating behavior.
Discussion
Studies have demonstrated the prevalence of TMP and
TTMP as sex pheromones in certain Tephritidae spe-
cies [22–24]. While the rectal Bacillus synthesis mecha-
nisms of TMP and TTMP have been further elucidated
[22], their olfactory recognition mechanisms remain
unexplored. Our study has identified Pbp5 as an odor-
ant binding protein that detects TMP and TTMP, thereby
paving the way for further investigation into the olfactory
recognition mechanism of these compounds. Given the
common occurrence of TMP and TTMP in Tephriti-
dae [22, 23], along with the high conservation of Pbp5
within this family, we hypothesize that the recognition
mechanisms for these compounds may be shared among
Tephritidae species. It is worth noting that Pbp5 is also
present in other dipteran insects such as Drosophila [26];
however, studies have revealed that Pbp5 is not required
for odorant transport in Drosophila [27]. erefore, it
cannot be ruled out that Pbp5 may also play additional
roles in the olfactory process of B. dorsalis. Furthermore,
the circadian influence on odorant receptors (Ors), which
play a direct role in neuronal activation either inde-
pendently or in conjunction with other factors such as
Fig. 5 Influence of Tim on Pbp5 expression and female mating. a Tissue expression pattern of Tim in females (replicates = 5, F(3,16) = 70.45,
P < 0.0001, one-way ANOVA). b RNAi efficiency of Tim (replicates = 5, P = 0.0001, independent sample t test). c Relative expression of Pbp5 in female
antenna after Tim knockdown (replicates = 5, P < 0.0001, independent sample t test). d Influence of Tim knockdown on mated female number
(replicates = 5, P = 0.0004, paired sample t test). e Expression patterns of Tim and Pbp5 in the head and antenna of females with biological clock
reversed ( Tim: replicates = 4, F(5,18) = 5.199, P = 0.004, one-way ANOVA; Pbp5: replicates = 4, F(5,18) = 4.954, P = 0.005, one-way ANOVA). f Mated
number of females with biological clock reversed in the evening (replicates = 5, P < 0.0001, paired sample t test). g Mated number of females
with biological clock reversed in the morning (replicates = 5, P < 0.0001, paired sample t test)
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Jiaoetal. BMC Biology (2025) 23:56
odorant binding proteins, may also impact the percep-
tion of sex pheromones. Although the Ors identified in
the head transcriptome did not exhibit rhythmic expres-
sion, further research is needed to identify the specific
Ors responsible for detecting sex pheromones.
It is notable that certain phenolic compounds, such as
(E)-coniferyl alcohol (E-CF) and 2-allyl-4,5-dimethoxy-
phenol (DMP), are also considered to effectively enhance
the male attraction to females and increase their mating
success rate [28, 29]. Moreover, E-CF behaves in the same
manner as TMP and TTMP, exerting a higher attraction
to females at dusk [30]. However, both E-CF and DMP
are derived from methyl eugenol (ME), which is a plant
volatile [31]. E-CF and DMP can only be emitted after
males ingest ME [32]. Nevertheless, in the natural and
experimental populations, when males have no access to
ME, they can still carry out mating behavior effectively,
which might imply that these phenolic compounds can
enhance the males’ attraction to the females, but they are
not indispensable.
Similar to other behaviors, olfactory perception plays a
crucial role in guiding animal behavior, which can vary
according to the time of day [33]. Recent research has
demonstrated connections between the olfactory system
and the biological clock [34, 35]. e expression of Obps
and Ors, which are related to mating and foraging, may
exhibit a strong correlation with circadian changes in
environmental factors such as photoperiod and tempera-
ture [36, 37]. ese environmental factors have the ability
to modify olfactory recognition abilities. Our study also
revealed that B. dorsalis detects sex pheromones (TMP
and TTMP) in a circadian rhythm similar to beetles,
Drosophila, and mosquitoes [36–40]. Furthermore, our
research showed that Tim can influence the expression
of Pbp5, thereby controlling females’ sensitivity to sex
pheromones and impacting their diurnal mating behav-
ior. Tim is considered a core oscillatory gene in insect
brains [41, 42]; mutant flies lacking Tim demonstrate an
inability to respond to odors in a rhythmic manner, fur-
ther confirming regulation by the circadian clock [43].
However, it is still unclear how Tim affects the expression
of Pbp5, and the detailed regulatory pathway still needs
to be further elucidated.
Our study still has certain limitations. While our behav-
ioral and molecular experiments have confirmed that cir-
cadian mating behavior is influenced by changes in the
rhythm of recognition ability to sex pheromone, we have
only been able to identify the Obp responsible for sex
pheromone in the olfactory system, with the related Ors
remaining unknown. Additionally, while it has been dem-
onstrated that Tim may indirectly affect the expression of
Pbp5, the precise interaction mode between the rhythmic
system and the olfactory system remains unclear.
Conclusions
In recent years, significant advancements have been
achieved in the identification of the sex pheromone of
male B. dorsalis. Specifically, it has been shown that rec-
tal Bacillus of males utilizes glucose and amino acids as
precursors to produce sex pheromone-TMP and TTMP
[22–24]. However, there remains limited understand-
ing regarding how females perceive TMP and TTMP
released by males. is study demonstrates for the first
time that odorant binding protein-Pbp5 is responsible
for binding TMP and TTMP in female antennae. Fur-
thermore, it has been revealed that the rhythmic mat-
ing behavior of females is associated with the rhythmic
expression of Pbp5, which can be influenced by the clock
gene-Tim. Our research significantly contributes to our
comprehension of the rhythmic mating behavior exhib-
ited by B. dorsalis.
Methods
Insect rearing
e B. dorsalis strain is reared under laboratory condi-
tions (27 ± 1 °C, 12:12 h light:dark cycle, 70–80% RH).
e larva is fed by a maize-based artificial diet contain-
ing 150 g of corn flour, 150 g of banana, 0.6 g of sodium
benzoate, 30 g of yeast, 30 g of sucrose, 30 g of paper
towel, 1.2 mL of hydrochloric acid and 300 mL of water.
e adult is manually fed by solid diet (consisting of 50 g
yeast hydrolysate and 50 g sucrose) and sterile water in
daily.
Recording offemale mating frequency atvarious time
intervals throughouttheday
To record the mating numbers of mature females (15 days
old) at different times of day, 15 mature males and 15
mature females were placed in a 35 × 35 × 35 cm wooden
cage. e day was divided into three observation periods
(0:00–8:00, 8:00–16:00, 16:00–24:00), and the number
of mated females during each period was recorded indi-
vidually. Five replicate cages were observed for each time
period.
Females attracted bysex pheromones atdierent time
periods inaday
e attractiveness of sex pheromones (TMP and TTMP)
to mature females was assessed at various time inter-
vals throughout the day. Briefly, 100 ml of TMP and
TTMP, diluted in ethanol (TMP: 2 mg/mL, TTMP: 1 mg/
mL), were placed in traps made of transparent plastic
vials (20 × 6 cm) sealed with a yellow lid featuring small
entrances for fly entry. e attraction assay was con-
ducted in a test chamber assembled with a ventilated
lid-covered plastic cylinder (120 × 30 cm). e day was
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Page 9 of 12
Jiaoetal. BMC Biology (2025) 23:56
divided into three observation periods (0:00–8:00, 8:00–
16:00, 16:00–24:00), and the number of females trapped
by the sex pheromone during each period was recorded
separately. Eight replicates were recorded for each period.
EAG response recording
EAG analysis was conducted to assess the electrogram
responses in the antenna of mature females exposed to
TMP and TTMP. For EAG preparations, the antenna of
a female was excised and positioned between two glass
electrodes (with one electrode connected to the antenna
tip). e antenna tip was gently trimmed to facilitate elec-
trical contact. Diluted solutions of TMP and/or TTMP
in ethanol were utilized as stimulants. Ten microliters of
sex pheromone (TMP: 2 mg/mL, TTMP: 1 mg/mL) were
applied onto the filter paper, which was then positioned
near the air inlet and stimulated five times. e signals
from the antennae were analyzed using GC-EAD 2014
software (version 4.6, Syntech). Solvent was used as a
negative control to stimulate each tested antenna in the
assays, and the resulting response values were recorded.
Subsequently, the response values were obtained by stim-
ulating the antennae with the compound dissolved in the
solvent. Finally, for statistical analysis, we calculated the
final result by subtracting the response value obtained
from stimulating the antennae with the compound dis-
solved in solvent from that obtained by stimulating them
with just solvent.
Transcriptome sequencing andgene identication
To identify the clock and olfactory genes that contribute
to female mating preference, the female RNA-seq was
done for mature female heads at different time periods
in a day (0:00, 4:00, 8:00, 12:00, 16:00, 20:00). Five rep-
licate samples were prepared for each period, with five
heads dissected for RNA extraction per sample. Subse-
quently, paired-end RNA-seq libraries were prepared and
sequenced on an Illumina HiSeq2000 platform. Briefly,
raw reads were generated in FASTQ format and sorted
by barcodes for further analysis. Prior to assembly, pre-
processing of paired-end raw reads from each cDNA
library was conducted to remove adapters, low-quality
sequences (Q < 20), and reads contaminated with micro-
bial sequences. Clean reads were then de novo assembled
to produce contigs. An index of the reference genome of
B. dorsalis was constructed, and paired-end clean reads
were mapped to the reference genome using HISAT2
2.4 with parameters including “-rna-strandness RF” and
defaults [44]. StringTie software was utilized to calculate
normalized gene expression values (FPKM) for evaluat-
ing transcript expression abundances [45]. Subsequently,
gene differential expression analysis was performed
using DESeq2 software [46]. Genes/transcripts with a
false discovery rate (FDR) below 0.01 and absolute fold
change ≥ 2 were considered DEGs. Principal compo-
nent analysis (PCA) was conducted using the R package
gmodels to elucidate the structure/relationship of the
samples. Pathway enrichment analysis was carried out to
identify clock genes in the circadian rhythm pathway.
Expression validation ofcandidate genes byqRT‑PCR
qRT-PCR analysis was used to validate gene expression
in antenna and other tissues. Total RNA in the tissues
of mature females was extracted using TRIzol reagent.
Subsequently, cDNA synthesis was conducted utiliz-
ing the One-Step gDNA Removal and cDNA Synthesis
SuperMix Kit (TransGen Biotech, Beijing, China). qRT-
PCR was then performed using the PerfectStarTM Green
qPCR SuperMix Kit (TransGen Biotech, Beijing, China)
to assess gene expression levels. Gene-specific primers
for the target genes were designed via primer blast on the
NCBI website (Additional file10: TableS2). Before con-
ducting qPCR, the amplification efficiency of the primers
was evaluated, and only primers with amplification effi-
ciency within the range of 90–110% were used for fur-
ther qPCR. e α-tubulin and actin genes were utilized
as reference genes [47]. PCR procedures were conducted
following the manufacturer’s instructions. e three-step
PCR procedures and melt curve analysis were carried out
in accordance with the manufacturer’s instructions. Spe-
cifically, the qPCR protocol included an initial denatura-
tion at 95 ℃ for 30 s, followed by 40 cycles of PCR with
denaturation at 95 ℃ for 5 s, annealing at 55℃ for 30 s,
and extension at 72 ℃ for 30 s. e protocol concluded
with a step to generate a melt curve, which involved an
initial denaturation at 95 ℃ for 15 s, annealing at 60 ℃
for 30 s, and a final denaturation at 95 °C for another 15
s. Only experiments resulting in a single peak on the melt
curve were considered as valid amplification reactions.
Subsequently, data normalization was performed using
the method of 2−ΔΔCt.
Phylogenetic sequence analysis
Phylogenetic analysis was conducted using amino
acid sequence alignments for Obp sequences identi-
fied from insect genomes. Specifically, Pbp5 amino
acid sequence was used to do protein-to-protein blast
in NCBI. en the top 10 most similar Obp sequences
(accession number: XP_011198820.1, XP_039959984.1,
XP_011193147.1, XP_004525016.1, XP_023303590.2,
XP_037935942.1, XP_058983721.1, XP_013101515.1,
XP_020809323.1, and XP_002078516.2) in other Dip-
tera species were used to generate the phylogenetic tree.
Obp amino acid sequence analyses were performed with
MEGA11, and maximum likelihood (ML) tree recon-
struction was performed using the Poisson model and
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 12
Jiaoetal. BMC Biology (2025) 23:56
uniform rates [48]. e ML heuristic search was per-
formed with the nearest neighbor-change method, and
the initial tree was selected by applying the neighbor-
joining method to a matrix of pairwise distances esti-
mated using the JTT method. e accuracy of the tree
was tested with bootstrapping using 100 replicates. e
conservation of the Obp proteins was determined using
the WebLogo tool (https:// weblo go. berke ley. edu/ logo.
cgi).
RNA interference
Double-stranded RNA (dsRNA) primers, containing the
T7 promoter sequence, were designed utilizing the cod-
ing sequences (CDSs) of the target genes as templates
(Additional file 10: Table S2). e MEGAscript RNAi
Kit (ermo Fisher Scientific, USA) was employed for
the synthesis and purification of dsRNA following the
manufacturer’s instructions. e GFP gene (GenBank
accession number: AHE38523) served as the RNAi nega-
tive control. To induce a knockdown effect on the target
genes, 0.5 μL dsRNA (1000 ng/μL) was injected into the
abdomen. e knockdown efficiency of the genes was
assessed using qRT-PCR 24h after dsRNA injection.
Besides, the survival rate of 30 dsRNA-injected females
was also recorded.
Mating competition assay
Mating competition assays between females with dif-
ferent treatments were conducted in a wooden cage
(35 cm × 35 cm × 35 cm). Briefly, pronotum of females
injected with dsRNA of Obp99a (Pbp5) and GFP were
colored with different colors. en 30 females injected
dsRNA of Obp99a (Pbp5) and 30 females injected dsRNA
of GFP were introduced into the wooden cage, in which
30 mature unmated males were placed. en the mated
female number was recorded during 20:00 to 22:00. Each
assay was replicated five times.
For mating ability comparison between females with
changed biological clocks and the controls, the same
assays were done as the above methods.
Protein–ligand docking simulation
After homologous protein modeling was conducted
using the Swiss-Model software, models with high scores
were selected for subsequent model validation [49]. e
quality of the Pbp5 model was assessed by Verify-3D, and
ERRAT, within the SAVES V7.0 software [50]. TMP and
TTMP models were downloaded from the NCBI web-
site. Protein–ligand docking simulations were performed
using Autodock Vina [51]. e binding result with the
lowest binding energy was selected, processed using
PyMOL [52], and further analyzed and visualized using
ProteinPlus [53] and Protein–Ligand Interaction Profiler
[54].
Binding ability ofPbp5 toTMP andTTMP
e recombinant Pbp5 protein was primarily obtained
through in vitro expression in Escherichia coli Rosetta
(DE3) cells, following a previously established protocol
[55]. In brief, PCR primers with restriction sites were
designed according to the CDS of Pbp5. Subsequently,
PCR products were purified and ligated to the pet-sumo
prokaryotic expression vector. e resulting vector was
then transformed into Escherichia coli Rosetta (DE3)
cells for expression. Positive clones were selected based
on kanamycin resistance, and their sequences were
confirmed through sequencing to ensure the correct
sequence. Verified clones were cultured in LB medium
supplemented with kanamycin at 37 ℃ for 16 h. Subse-
quently, 100 ml of bacterial culture was inoculated into
an LB medium containing 0.1 mM IPTG and incubated
at 18 ℃ for 8 h. e bacteria were then harvested by cen-
trifugation at 8000 rpm and resuspended in lysis buffer
(80 mM Tris–HCl, 200 mM NaCl, 1 mM EDTA, 4% glyc-
erol, pH 7.2, 0.5 mM PMSF). Sonication (3 s, five passes)
was performed to lyse the bacterial cells. e recombi-
nant proteins present in the supernatant were collected
by centrifugation. Subsequently, the proteins were puri-
fied by two rounds of anion-exchange chromatography
and concentrated using an ultrafiltration cube. e purity
of the purified recombinant proteins was confirmed by
SDS-PAGE analysis. Another recombinant protein Pbp5-
mutant, whose binding sites for TMP and TTMP were
mutated, was also prepared with the method above. Spe-
cifically, according to the docking results, the binding
sites (Ile-28, Phe-31, Phe-95, and Phe-147) (Additional
file10: TableS1) of Pbp5 were mutated to alanine.
en the fluorescence binding assay using 1-NPN
with Pbp was conducted on a Microplate Reader (er-
moScientific Varioskan LUX), following a method pre-
viously described in locust studies [56]. e excitation
wavelength was set at 337 nm, and the emission wave-
length was set at 380–520 nm. Pbp5 or Pbp5-mutant (2
μM dissolved in 50 mM Tris–HCl, pH = 7.4) was mixed
with 1-NPN (2 μM-16 μM dissolved in chromatographic
methanol), and the maximum fluorescence intensity was
recorded. A nonlinear regression analysis for one-site-
specific binding was carried out based on the gradient
fluorescence intensity. e dissociation constant (Kd)
and the maximum fluorescence intensity (Bmax) for the
binding of 1-NPN to the protein were computed.
For competitive binding assays, various concentrations
of TMP or TTMP (dissolved in chromatographic meth-
anol) were incrementally added to the mixture of the
recombinant protein and 1-NPN (2 μM dissolved in 50
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 12
Jiaoetal. BMC Biology (2025) 23:56
mM Tris–HCl, pH = 7.4). Subsequently, the fluorescence
intensity at different concentrations of TMP or TTMP
was recorded. e inhibition constant (Ki) for TMP or
TTMP competing with 1-NPN was calculated based on
the fluorescence intensity. A lower Ki value indicates a
stronger affinity between the protein and TMP or TTMP.
Data analysis
Statistical analysis methods used in the study were indi-
cated in the figure legends. Differences were considered
significant when P < 0.05. All data were analyzed using
the GraphPad Prism version 10, GraphPad Software, La
Jolla, CA, USA, https:// www. graph pad. com/
Abbreviations
TMP Trimethylpyrazine
TTMP Tetramethylpyrazine
EAG Electroantennogram
Tim Timeless
DEGs Differentially expressed genes
Csps Chemosensory proteins
Obps Odorant binding proteins
Ors Odorant receptors
Pbp5 Pheromone binding protein 5
Obp99a Odorant binding protein 99a
1-NPN N-Phenyl-1-naphthylamine
Kd Dissociation constant
Ki Inhibition constant
E-CF (E)-coniferyl alcohol
DMP 2-Allyl-4,5-dimethoxyphenol
ME Methyl eugenol
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12915- 025- 02164-4.
Additional file 1. Fig. S1. Difference of head transcriptome at different time
of a day
Additional file 2. Fig. S2. RNAi of Pbp5 and Obp99a
Additional file 3. Fig. S3. Example traces of Pbp5 knocked down female
antenna responses to sex pheromone
Additional file 4. Fig. S4. Construction and evaluation of Pbp5 protein
model
Additional file 5. Influence of Tim and biological rhythm on female EAG
response to sex pheromone
Additional file 6. Dataset S1. Head gene expression profile. Dataset S2. DE
genes screening between head at 20:00 and 0:00
Additional file 7. Dataset S3 DE genes screening between head at 20:00
and 4:00. Dataset S4 DE genes screening between head at 20:00 and 8:00
Additional file 8. Dataset S5 DE genes screening between head at 20:00
and 12:00. Dataset S6 DE genes screening between head at 20:00 and
16:00
Additional file 9. Dataset S7 Expression of DEG in Circadian rhythm path-
way. Dataset S8 Expression of olfactory genes
Additional file 10. Tables S1-S2. Table S1 Predication of binding site amino
acid residue of Pbp5. Table S2 Primers used in the study
Acknowledgements
We thank Guangzhou Genedenovo Biotechnology Co., Ltd., for assisting in
sequencing.
Authors’ contributions
D.C. and Y.J. conceived and designed the project. Y.J. and G.L. performed
experiments and analyzed data. D.C. made the graphs. D.C. and Y.J. wrote the
manuscript. D.C. and Y.L. provided the research platform. All authors read and
approved the final manuscript.
Funding
The study was funded by the National Natural Science Foundation of China
(No. 32372520 and No. 3212200346) and the National Key Research and
Development Program of China (No. 2023YFD1401401-08).
Data availability
All the data needed to evaluate the conclusions in the paper are presented
in the paper and/or the Supplementary Materials. The accession number
of Tim, Pbp5 and Obp99a are XM_011215785.4, XM_011200518.4 and
XM_011206554.3, respectively. RNA-sequencing data have been deposited in
the Genome Sequence Read Archive Database of the National Genomics Data
Center (CRA022721).
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 19 August 2024 Accepted: 17 February 2025
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