A timeless gene-regulated pheromone binding protein governs female mating behavior in Bactrocera dorsalis

doi:10.21203/rs.3.rs-4935346/v1

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A timeless gene-regulated pheromone binding protein governs female mating behavior in Bactrocera dorsalis

https://doi.org/10.21203/rs.3.rs-4935346/v1

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The rhythmic mating behavior of insects has been extensively documented, yet the regulation of this behavior through sex pheromone sensing olfactory genes controlled by the clock genes in the rhythm pathway remains unclear. 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 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 regulated by Tim. Furthermore, manipulation of female circadian rhythm resulted in increased morning 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. Our results also contribute to a better understanding of the molecular mechanisms underlying rhythmic insect mating behavior.

Reproductive behavior, including courtship, mating, and oviposition, is a fundamental biological activity for insects to regulate their populations¹. Mating behavior serves as the pivotal step in reproductive activity² and is typically initiated following successful courtship³. 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 sex⁷.

Environmental factors exert a significant influence on the mating behavior of insects⁸. The periodic fluctuations 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 temperature variations, represent important entrainment factors affecting insect mating behavior¹⁰. The circadian rhythm is an endogenous biological process driven by molecular oscillations of the internal clock¹¹. Most central clocks undergo endogenous cyclic oscillations 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 pheromone-mediated courtship behaviors^17–19. Circadian rhythms governing mating behavior play a pivotal role in population survival and propagation among animals²⁰. This behavioral adaptation enables animals to evade adverse environmental conditions such as predators and concentrate reproductive activities at opportune times based on their ecological niches, thereby enhancing safe reproduction and promoting stable population growth²¹. A comprehensive understanding of how and why animals mate at specific times provides valuable insights into their reproductive strategies, evolutionary adaptations, and ecological interactions, 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. Electroantennogram (EAG) recordings and behavioral assays indicate a rhythmic variation in female sensitivity to sex pheromone, peaking in the evening. Comparative head transcriptome analysis reveals that the sex pheromone binding protein-Pbp5 is responsible for mediating female perception of sex pheromone. Furthermore, we show that the clock gene-Timeless (Tim) can regulate the rhythmic expression of Pbp5. This study contributes to our understanding of insect reproductive behavior and serves as an important supplement to research on insect olfactory perception.

Mating behavior of females and their attraction to sex pheromone peaking in the evening

To investigate whether female mating occurs at specific times of the day, we monitored mating frequency throughout the day. The 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 differences 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 morning (Fig. 1c-1e, Figure S1a-1c). These results suggest a rhythmic pattern in female mating behavior over a daily cycle, potentially regulated by rhythmic expression of clock genes and olfactory genes.

Screening for genes associated with rhythmic mating behavior

To further screen and identify the olfactory and clock genes that regulate rhythmic mating of females, we conducted female head RNA-seq analysis at 4-hour intervals throughout the day. The results revealed no significant differences in head gene expression patterns at different times of the day (Figure S2a, Dataset S1). However, an increase in the time difference for female head sampling led to a greater number of identified differentially expressed genes (DEGs) (Fig. 2a, Figure S2b-2f and Datasets S2-S6). Subsequently, KEGG analysis was performed on the most DEGs screened between 8:00 and 20:00 to identify genes responsible for regulating rhythmic mating behavior in females.

The 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 2e, Dataset S7). Given that olfactory genes, such as chemosensory proteins (Csps), odorant binding proteins (Obps), and odorant receptors (Ors), are primarily responsible for binding volatiles, including sex pheromones²⁵, we analyzed the expression patterns of olfactory genes in female heads at different times to identify potential genes involved in sex pheromone binding. The results showed differential expression of various olfactory genes between heads at 8:00 and 20:00 (Fig. 2d, Dataset S8). Among these genes, pheromone binding protein 5 (Pbp5) exhibited the highest and lowest 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). These findings suggest that peak mating behavior among females at 20:00 may be associated with elevated levels of Tim and Pbp5.

Pbp5 exhibits robust binding capabilities to both TMP and TTMP

The binding abilities of Pbp5 to N-phenyl-1-naphthylamine (1-NPN), TMP and TTMP were assessed using Alphafold 2.0 for homology modeling of Pbp5, followed by prediction of the binding sites with Autodock Vina. The evaluation results indicated a high-quality score of 96.7 for the constructed Pbp5 model (Figure S3b), and all amino acids fell within the optimal and suboptimal regions in the Ramachandran Plot (Figure S3c), suggesting suitability for docking analysis. The docking results revealed that Phe-31, Ile-52, Lys-55, Phe-95, and Phe-147 constituted the binding site of Pbp5 to 1-NPN (Fig. 3a, Table S1). 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 3c, Table S1). Furthermore, it was found that Phe-31, Phe-95, and Phe-147 were common binding sites for TMP, TTMP and 1-NPN to Pbp5 (Table S1), suggesting that competitive fluorescence binding experiments 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 Kd value of 10.57 µM (Fig. 3d and 3e). Moreover, competitive fluorescence binding experiments indicated that both TMP and TTMP exhibited stronger affinities towards Pbp5 compared to 1-NPN with Kd values of 0.027µM and 0.0027µM, respectively (Fig. 3f and 3g). These findings suggest that the perception of TMP and TTMP by Pbp5 may regulate the rhythmic mating behavior of females.

RNAi-mediated knockdown of Pbp5 results in impaired female mating and reduced EAG response to TMP and TTMP

We then conducted 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 Diptera species revealed that Pbp5 was highly conserved within Tephritidae (Fig. 4a). The 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 exhibited the highest expression in female antennae, while Obp99a peaked in the head of females (Fig. 4c and 4d). Subsequent RNAi experiments resulted in a decrease of more than 50% in the expression levels of Pbp5 and Obp99a (Figure S4a and 4b), with no significant impact on survival observed (Figure S4c and 4d). 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 4f). Additionally, EAG responses to TMP, TTMP, and mixture of TMP and TTMP were significantly reduced in females with knocked down Pbp5 expression (Fig. 4g-4i, Figure S5a-5c). These findings suggest that Pbp5 plays a role in female mating and response to sex pheromone-TMP and TTMP.

Tim modulates the expression of Pbp5

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. The 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. 5b and 5c). Additionally, knockdown of Tim significantly reduced female mating frequency (Fig. 5d) and EAG response to sex pheromone (Figure S6a-6c). 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 2f), with both genes exhibiting peak expressions at 8:00. Mating competition assays also indicated that fewer females with reversed circadian rhythms mated with males than control 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 (Figure S6d-6i). These findings collectively suggest that Tim can modulate both Pbp5 expression and female mating behavior.

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 rectal Bacillus of male utilizes glucose and amino acids as precursors to produce sex pheromone-TMP and TTMP^22–24. However, there remains limited understanding regarding how females perceive TMP and TTMP released by males. This study demonstrates for the first time that odorant binding protein-Pbp5 is responsible for binding TMP and TTMP in female antennae. Furthermore, it has been revealed that the rhythmic mating 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 exhibited by B. dorsalis.

Studies have demonstrated the prevalence of TMP and TTMP as sex pheromones in certain Tephritidae species^22–24. While the rectal Bacillus synthesis mechanisms of TMP and TTMP have been further elucidated²², their olfactory recognition mechanisms remain unexplored. Our study has identified Pbp5 as an odorant binding protein that recognizes 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 Tephritidae, 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 Drosophila²⁶; however, studies have revealed differing roles for Pbp5 in Drosophila²⁷. Therefore, it cannot be ruled out that Pbp5 may also play additional roles in the olfactory process of B. dorsalis.

Similar to other behaviors, olfactory perception plays a crucial role in guiding animal behavior, which can vary according to the time of day²⁸. Recent research has demonstrated connections between the olfactory system and the biological clock^29,30. The 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 temperature^31,32. These environmental factors have the ability to modify olfactory recognition abilities. Our study also revealed that B. dorsalis detects sex pheromone (TMP and TTMP) in a circadian rhythm similar to Drosophila and mosquitoes^33–35. Furthermore, our research showed that Tim can influence the expression of Pbp5, thereby controlling females' sensitivity to sex pheromone and impacting their diurnal mating behavior. Tim is considered a core oscillatory gene in insect brains^36,37; mutant flies lacking Tim demonstrate an inability to respond to odors in a rhythmic manner, further confirming regulation by the circadian clock³⁸.

However, our study still has certain limitations. While our behavioral and molecular experiments have confirmed that circadian 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 demonstrated that Tim indirectly affects the expression of Pbp5, the precise interaction mode between the rhythmic system and the olfactory system remains unclear.

Insect rearing

The B. dorsalis strain is reared under laboratory condition (27 ± 1°C, 12:12 h light:dark cycle, 70–80% RH). The larva is fed by a maize-based artificial diet containing 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. The adult is manually fed by solid diet (consisting of 50 g yeast hydrolysate and 50 g sucrose) and sterile water in daily.

Recording of female mating frequency at various time intervals throughout the day

To record the mating numbers of mature females (15 days old) at different time of day, 15 mature males and 15 mature females were placed in a 35× 35×35 cm wooden cage. The 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 individually. Five replicate cages were observed for each time period.

Females attracted by sex pheromone at different time periods in a day

The attractiveness of sex pheromones (TMP and TTMP) to mature females was assessed at various time intervals 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 x 6 cm) sealed with a yellow lid featuring small entrances for fly entry. The attraction assay was conducted in a test chamber assembled with a ventilated lid-covered plastic cylinder (120 x 30 cm). The day was 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). The antenna tip was gently trimmed to facilitate electrical 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. The signals from the antennae were analyzed using GC-EAD 2014 software (version 4.6, Syntech).

Transcriptome sequencing and gene identification

To identify the clock and olfactory genes that contribute to female mating preference, the female RNA-seq was done for mature female head at different time periods in a day (0:00, 4:00, 8:00, 12:00, 16:00, 20:00). Five replicate samples were prepared for each period, with five heads dissected for RNA extraction per sample. Subsequently, 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, preprocessing of paired-end raw reads from each cDNA library was conducted to remove adapters, low-quality sequences (Q < 20), and reads contaminated with microbial 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. StringTie software was utilized to calculate normalized gene expression values (FPKM) for evaluating transcript expression abundances. Subsequently, gene differential expression analysis was performed using DESeq2 software. Genes/transcripts with a false discovery rate (FDR) below 0.01 and absolute fold change ≥ 2 were considered DEGs. Principal component 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 circadian rhythm pathway. Expression validation of candidate genes by qRT-PCR

qRT-PCR analysis was used to validate gene expression in antenna and other tissues. Total RNA in the tissues of mature female was extracted using TRIzol reagent. Subsequently, cDNA synthesis was conducted utilizing 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 (Table S2). The α-tubulin and actin genes were utilized as reference genes³⁹. PCR procedures were conducted following the manufacturer's instructions.

Phylogenetic sequence analysis

Phylogenetic analysis was conducted using amino acid sequence alignments for Obp sequences identified from insect genomes. Obp amino acid sequence analyses were performed with MEGA11, and maximum likelihood (ML) tree reconstruction was performed using the Poisson model and uniform rates. The ML heuristic search was performed with the nearest neighbor-change method, and the initial tree was selected by applying the neighbor-joining method to a matrix of pairwise distances estimated using the JTT method. The accuracy of the tree was tested with bootstrapping using 100 replicates. The conservation of the Obp proteins was determined using the WebLogo tool (https://weblogo.berkeley.edu/logo.cgi).

RNA interference

Double-stranded RNA (dsRNA) primers, containing the T7 promoter sequence, were designed utilizing the coding sequences (CDSs) of the target genes as templates (Table S2). The MEGAscript RNAi Kit (Thermo Fisher Scientific, United States) was employed for the synthesis and purification of dsRNA following the manufacturer's instructions. The GFP gene (GenBank accession number: AHE38523) served as the RNAi negative control. To induce knockdown effect to the target genes, 0.5 µL dsRNA (1000ng/µL) was injected into the abdomen. The 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 different treatments were conducted in a wooden cage (35 cm×35 cm×35 cm). Briefly, pronota of females injected with dsRNA of Obp99a (Pbp5) and GFP were colored with different colors. Then 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. Then the mated female number was recorded during 20:00 to 22:00. Each assay was replicated five times.

For mating ability comparison between female with changed biological clocks and the controls. The same assays were done as the above methods.

Binding ability of Pbp5 to TMP and TTMP

The recombinant Pbp5 protein was primarily obtained through in vitro expression in Escherichia coli Rosetta (DE3) cells, following a previously established protocol⁴⁰. 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. The 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 hours. Subsequently, 100 ml of bacterial culture was inoculated into LB medium containing 0.1 mM IPTG and incubated at 18℃ for 8 hours. The bacteria were then harvested by centrifugation at 8000 rpm and resuspended in lysis buffer (80 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 4% glycerol, pH 7.2, 0.5 mM PMSF). Sonication (3 s, five passes) was performed to lyse the bacterial cells. The recombinant proteins present in the supernatant were collected by centrifugation. Subsequently, the proteins were purified by two rounds of anion-exchange chromatography and concentrated using an ultrafiltration cube. The purity of the purified recombinant proteins was confirmed by SDS-PAGE analysis.

The fluorescence binding assay using 1-NPN with Pbp was conducted on a Microplate Reader (ThermoScientific Varioskan LUX), following a method previously described in locust studies⁴¹. The excitation wavelength was set at 337 nm, and the emission wavelength was set at 380–520 nm. Pbp (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. The dissociation constant of 1-NPN binding to Pbp was calculated using the one-site specific binding method in GraphPad 8.0 software. Subsequently, dissociation constants for Pbp5 were determined. For competitive binding assays, TMP and TTMP (0, 4, 8, 12, 16 µM dissolved in chromatographic methanol) were used as competitors to bind Pbp in the Pbp/1-NPN complex.

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. The quality of the Pbp5 model was assessed by Verify-3D, and ERRAT, within the SAVES V7.0 software⁴². TMP and TTMP models were downloaded from the NCBI website. Protein-ligand docking simulations were performed using Autodock Vina. The binding result with the lowest binding energy was selected, processed using PyMOL, and further analyzed and visualized using ProteinPlus and Protein-Ligand Interaction Profiler.

Data analysis

Statistical analysis methods used in the study were indicated 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.graphpad.com/.

Competing interests

The authors declare no competing interests.

Author Contribution

Y.J and G.L finish the experiment. Y.L gave valuable advice. D.C and Y.J wrote the main manuscript text and prepared figures . All authors reviewed the manuscript.

Acknowledgements

We thank Guangzhou Genedenovo Biotechnology Co., Ltd., for assisting in sequencing. We are grateful to 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) for funding support.

Data Availability

Sequence data that support the findings of this study have been deposited in the CNCB with the primary accession code PRJWB13140

Wade, M. J. THE EVOLUTION OF INSECT MATING SYSTEMS. Evolution; international journal of organic evolution 38, 706–708, (1984).
Evans, H. E. Insect behavior. Science (New York, N.Y.) 194, 612–613, (1976).
Dixson, A. Mating systems and strategies. American Journal of Human Biology 17, 390–391, (2005).
Slessor, K. N., Winston, M. L. & Le Conte, Y. Pheromone communication in the honeybee (Apis mellifera L.). Journal of Chemical Ecology 31, 2731–2745, (2005).
Woods, W. A., Jr., Hendrickson, H., Mason, J. & Lewis, S. M. Energy and predation costs of firefly courtship signals. American Naturalist 170, 702–708, (2007).
Wagner, Jr. & Reiser. The importance of calling song and courtship song in female mate choice in the variable field cricket. Animal behaviour 59, 1219–1226, (2000).
Spieth, H. T. Courtship behavior in Drosophila. Annual review of entomology 19, 385–405, (1974).
Saunders, D. S. The biological clock of insects. Scientific American 234, 114–121, (1976).
Soma, M. Behavioral and Evolutionary Perspectives on Visual Lateralization in Mating Birds: A Short Systematic Review. Frontiers in Physiology 12, (2022).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, 884–890, (2018).
Zhang, S. L., Yue, Z., Arnold, D. M., Artiushin, G. & Sehgal, A. A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux. Cell 173, 130-+, (2018).
Ito, C. & Tomioka, K. Heterogeneity of the Peripheral Circadian Systems in Drosophila melanogaster: A Review. Frontiers in Physiology 7, (2016).
Yao, Z. & Shafer, O. T. The Drosophila Circadian Clock Is a Variably Coupled Network of Multiple Peptidergic Units. Science 343, 1516–1520, (2014).
Mazuski, C. et al. Entrainment of Circadian Rhythms Depends on Firing Rates and Neuropeptide Release of VIP SCN Neurons. Neuron 99, 555-+, (2018).
Mohawk, J. A., Green, C. B. & Takahashi, J. S. in Annual Review of Neuroscience, Vol 35 Vol. 35 Annual Review of Neuroscience (ed S. E. Hyman) 445–462 (2012).
Tomioka, K. & Matsumoto, A. A comparative view of insect circadian clock systems. Cellular and Molecular Life Sciences 67, 1397–1406, (2010).
Miyamoto, T. & Amrein, H. Suppression of male courtship by a Drosophila pheromone receptor. Nature Neuroscience 11, 874–876, (2008).
Clowney, E. J., Iguchi, S., Bussell, J. J., Scheer, E. & Ruta, V. Multimodal Chemosensory Circuits Controlling Male Courtship in Drosophila. Neuron 87, 1036–1049, (2015).
Wada-Katsumata, A. & Schal, C. Antennal grooming facilitates courtship performance in a group-living insect, the German cockroach Blattella germanica. Scientific Reports 9, (2019).
Pittendrigh, C. S. Temporal organization: reflections of a Darwinian clock-watcher. Annual review of physiology 55, 16–54, (1993).
Vaz Nunes, M. & Saunders, D. Photoperiodic time measurement in insects: a review of clock models. Journal of biological rhythms 14, 84–104, (1999).
Ren, L., Ma, Y., Xie, M., Lu, Y. & Cheng, D. Rectal bacteria produce sex pheromones in the male oriental fruit fly. Current Biology 31, 2220-+, (2021).
Gao, Z. et al. Differences in rectal amino acid levels determine bacteria-originated sex pheromone specificity in two closely related flies. Isme Journal 17, 1741–1750, (2023).
Gui, S. Y., Yuval, B., Engl, T., Lu, Y. Y. & Cheng, D. F. Protein feeding mediates sex pheromone biosynthesis in an insect. Elife 12, (2023).
Ha, T. S. & Smith, D. P. Recent Insights into Insect Olfactory Receptors and Odorant-Binding Proteins. Insects 13, (2022).
Anholt, R. R. H. & Williams, T. I. The Soluble Proteome of the Drosophila Antenna. Chemical Senses 35, 21–30, (2010).
Larter, N. K., Sun, J. S. & Carlson, J. R. Organization and function of Drosophila odorant binding proteins. Elife 5, (2016).
Gadenne, C., Barrozo, R. B. & Anton, S. in Annual Review of Entomology, Vol 61 Vol. 61 Annual Review of Entomology (ed M. R. Berenbaum) 317–333 (2016).
Page, T. L. & Koelling, E. Circadian rhythm in olfactory response in the antennae controlled by the optic lobe in the cockroach. Journal of Insect Physiology 49, 697–707, (2003).
Decker, S., McConnaughey, S. & Page, T. L. Circadian regulation of insect olfactory learning. Proceedings of the National Academy of Sciences of the United States of America 104, 15905–15910, (2007).
Wang, Y. et al. Circabidian rhythm of sex pheromone reception in a scarab beetle. Current Biology 34, (2024).
Siciliano, P. et al. Sniffing Out Chemosensory Genes from the Mediterranean Fruit Fly, Ceratitis capitata. Plos One 9, (2014).
Das, S. & Dimopoulos, G. Molecular analysis of light pulse stimulated blood feeding inhibition in Anopheles gambiae. American Journal of Tropical Medicine and Hygiene 79, 333–333, (2008).
Wang, G. et al. Clock genes and environmental cues coordinate Anopheles pheromone synthesis, swarming, and mating. Science 371, 411-+, (2021).
Tanoue, S., Krishnan, P., Krishnan, B., Dryer, S. E. & Hardin, P. E. Circadian clocks in antennal neurons are necessary and sufficient for olfaction rhythms in Drosophila. Current Biology 14, 638–649, (2004).
Shafer, O. T. et al. Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes. Elife 11, (2022).
Ma, D. et al. Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation. Proceedings of the National Academy of Sciences of the United States of America 121, e2321338121-e2321338121, (2024).
Krishnan, B., Dryer, S. E. & Hardin, P. E. Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature 400, 375–378, (1999).
Shen, G.-M., Jiang, H.-B., Wang, X.-N. & Wang, J.-J. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). Bmc Molecular Biology 11, (2010).
Tian, Z. et al. Molecular characterization and functional analysis of pheromone binding proteins and general odorant binding proteins from Carposina sasakii Matsumura (Lepidoptera: Carposinidae). Pest Management Science 75, 234–245, (2019).
Ban, L. et al. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria. Cellular and Molecular Life Sciences 60, 390–400, (2003).
Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. Journal of biomolecular NMR 8, 477–486, (1996).

No competing interests reported.

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A timeless gene-regulated pheromone binding protein governs female mating behavior in Bactrocera dorsalis

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Version 1

Abstract

Figures

Introduction

Results

Mating behavior of females and their attraction to sex pheromone peaking in the evening

Screening for genes associated with rhythmic mating behavior

Pbp5 exhibits robust binding capabilities to both TMP and TTMP

Tim modulates the expression of Pbp5

Discussion

Materials and methods

Insect rearing

Recording of female mating frequency at various time intervals throughout the day

Females attracted by sex pheromone at different time periods in a day

EAG response recording

Transcriptome sequencing and gene identification

Phylogenetic sequence analysis

RNA interference

Mating competition assay

Binding ability of Pbp5 to TMP and TTMP

Protein-ligand docking simulation

Data analysis

Declarations

Competing interests

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1