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. 2015 Jan 14;5:7777. doi: 10.1038/srep07777

Gr33a Modulates Drosophila Male Courtship Preference

Yujia Hu 1, Yi Han 1, Yingyao Shao 1, Xingjun Wang 1, Yeqing Ma 1, Erjun Ling 2, Lei Xue 1,a
PMCID: PMC4648378  PMID: 25586066

Abstract

In any gamogenetic species, attraction between individuals of the opposite sex promotes reproductive success that guarantees their thriving. Consequently, mate determination between two sexes is effortless for an animal. However, choosing a spouse from numerous attractive partners of the opposite sex needs deliberation. In Drosophila melanogaster, both younger virgin females and older ones are equally liked options to males; nevertheless, when given options, males prefer younger females to older ones. Non-volatile cuticular hydrocarbons, considered as major pheromones in Drosophila, constitute females' sexual attraction that act through males' gustatory receptors (Grs) to elicit male courtship. To date, only a few putative Grs are known to play roles in male courtship. Here we report that loss of Gr33a function or abrogating the activity of Gr33a neurons does not disrupt male-female courtship, but eliminates males' preference for younger mates. Furthermore, ectopic expression of human amyloid precursor protein (APP) in Gr33a neurons abolishes males' preference behavior. Such function of APP is mediated by the transcription factor forkhead box O (dFoxO). These results not only provide mechanistic insights into Drosophila male courtship preference, but also establish a novel Drosophila model for Alzheimer's disease (AD).

To avoid futile reproductive efforts, an animal must distinguish conspecifics from other species and differentiate the sex of conspecific partners. It must also determine the most suitable mates from large amounts of available partners in order to maximize reproductive efficiency. Evolution endows Drosophila melanogaster males with the instinct to discriminate conspecifics from other Drosophila species1 and to discern females from males2. It also bestows on them the ability to select the most favorable mates among masses of desirable virgin females3.

In Drosophila, non-volatile cuticular hydrocarbons (CHCs) have been recognized as a type of major sex pheromone4,5, which convey information of an individual such as species and sex. Female-specific CHCs are detected by male gustatory receptors (Grs), a chemosensory receptor family mainly responsible for detecting non-volatile chemicals6, during tapping and licking steps in stereotypical male courtship behavior6,7,8,9. So far, only a few Grs, including Gr32a, Gr33a and Gr39a are reported to be engaged in Drosophila male courtship behavior10. While Gr32a acts to assist males to discriminate conspecifics from other species11, Gr39a is required for males to distinguish females from males12. On the other hand, Gr33a functions to inhibit male-male courtship13. Despite these findings, roles of the Grs in males' choices for the most favorable mates have remained largely unknown.

Our previous study sets a paradigm of choice model in which both options (younger virgin females and older ones) are proved to be attractive to Drosophila males, but males still intensely prefer younger mates to older ones3. Using this model, we explored the mechanisms by which males bias their potential mates. Gene loss-of-function, gain-of-function, and cell-inactivation experiments demonstrated that Gr33a and Gr33a neurons are essential for males' preference for younger mates. Since our previous data indicated that pan-neuronal expression of human amyloid precursor protein (APP) ablates males' preference for younger mates3, we sought to investigate whether APP expression in Gr33a neurons would affect this behavior. Indeed, we found that Gr33a neurons-specific expression of APP abolished males' preference for younger mates, and this function of APP is mediated by the transcription factor forkhead box O (dFoxO).

Methods

Drosophila Strains

Oregon R, w1118, w1, Gr39ak05106, Gr33aGal4, Gr33a1, UAS-Gr33a, UAS-APPΔCT, UAS-GFP and UAS-APP were obtained from Bloomington Drosophila Stock Center. Gr32aMI04430 was a gift from Dr. Zuoren Wang. Δppk23 and Δppk29 were gifts from Dr. Kristin Scott. UAS-TNT and UAS-TNTin were gifts from Dr. Aike Guo. dfoxOΔ94 was generously provided by Dr. Linda Partridge. Wild type (WT) controls for the mutants (Gr32a, Gr33a, Gr39a, ppk23, ppk29) were from the corresponding background strains.

Fly Rearing

Drosophila stocks were maintained on a standard corn flour, yeast and agar medium under a 12 h light and 12 h dark cycle at 25°C. Naive male and virgin female flies were collected at eclosion. Males were kept individually for three days before test. Females were housed in groups of 10 per vial and were transferred every three days to new vials containing fresh medium.

Courtship Behavior Assays

Courtship choice assays were performed as described before3. All courtship choice assays were carried out at approximately the same time each day (within 1 hour at the beginning of the 12 h illumination half of the cycle)14 by pairing a naive male together with two younger wild type virgin females and two older ones. Before tests, males were checked and unhealthy ones were excluded from experiments. All tests were recorded for 10 min with an HDR-CX270 digital video camera (Sony). After recording, videos were analyzed by a researcher who was blinded to the genotypes of males or age markers of females, using Noldus EthoVision® XT software (Noldus Information Technology). Given that mating success terminated male courtship behavior and that a successful mating was consensual, thus, either males' or females' choices could bias the mating results15. Tests in which male mated successfully within 10 min were excluded from analysis. The courtship index (CI) was calculated as the percentage of time that a male displayed any of the courtship steps towards the females during the 10 min observation period. In courtship choice assays, CIy or CIo represents CI towards younger virgin females or older ones, respectively; the total courtship index (CIt) represents a male's total CIs towards both younger virgin females and older ones in a choice assay. We also defined a preference index (PI): the relative difference between males' courtship percentage towards younger females and that towards older ones in a choice assay.

CHC Extraction

Cuticular hydrocarbon (CHC) extracts were obtained from 3-day (younger) and 30-day (older) old virgin females. Five replicate CHC samples were prepared for each age. For each sample, extract was obtained from five individuals, selected from a vial containing 10 flies. Flies were anesthetized with CO2, and were placed into an individual glass vial with 30 μl of hexane (Merck KGaA) containing 100 ng/μl of octadecane (C18) and 100 ng/μl of hexacosane (nC26) as injection standards. To achieve efficient extraction, the glass vial was gently agitated for 5 min16. The extract was removed and placed in a clean glass vial and stored at −20°C prior to analysis. Extracts were examined by gas chromatography and mass spectrometry (See Supplemental Experimental Procedures).

Statistical Analysis

Since CIs were generally not normally distributed, nonparametric tests were employed in statistical analysis. Comparisons of intragroup CIs (CIy and CIo) in courtship choice assays used the Related-samples Wilcoxon Signed Rank test. CIts and PIs in choice assays were compared using the Kruskal-Wallis test followed by the post-hoc Dunn's test. Besides, CIts and PIs in choice assays to test the roles of Grs in males' interest on females and in cell-inactivation experiments were compared using the Mann-Whitney U test. In analysis of GC and MS results, Mann-Whitney U test was also applied due to the small volume of samples.

Results

The roles of Grs in males' courtship preference behavior

To investigate the possible function of Gr32a, Gr33a and Gr39a in males' preference for younger mates, we employed their mutant alleles: Gr32aMI04430, Gr39ak05106 and Gr33aGal4 (a portion of the Gr33a coding region is replaced by Gal4 using homologous recombination13), and introduced these males into courtship choice assays. In courtship choice behavior, a prerequisite of surveying a male's preference for younger or older mates is that the male does not lose the sexual interest in females. The courtship intensity of a male is a good indicator of its sexual interest in females17. Accordingly, choices of males displaying drastically impaired courtship might be unrepresentative. To assess a male's sexual interest in females, we defined a total courtship index (CIt), which represents a male's courtship intensity towards both mates (See methods). CIt of Gr32aMI04430 males shows no significant difference from that of wild type controls (Figure 1A), indicating that loss of Gr32a does not affect male-female courtship. Likewise, CIt of Gr33aGal4 males is similar to that of wild type controls (Figure 1B), inferring that malfunction of Gr33a does not disrupt male-female courtship. In contrast, mutation in Gr39a (Gr39ak05106) results in significantly reduced courtship intensity (Figure 1C), consistent with its reported role in male-female courtship12. In addition to Grs, chemosensory receptors such as degenerin/epithelial sodium channel/pickpocket (Ppk) family, also modulate male courtship behavior18. Therefore, we checked Δppk23 and Δppk29 males, which are loss-of-function mutants of ppk23 and ppk29 genes that are known to play critical roles in male courtship behavior19,20. We found that CIt of Δppk23 males decreases remarkably (Figure S1A), consistent with its role in male-female courtship20. Intriguingly, Δppk29 males displayed similar CIt as that of wild type control (Figure S1B), implying that dysfunction of Ppk29 does not significantly reduce male-female courtship intensity.

Figure 1. The roles of Grs in males' courtship preference behavior.

Figure 1

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(A) Total courtship indices of wild type males and Gr32a mutant males in courtship choice assays. Mean ± standard error of the mean (SEM), n = 21 for wild type males and 24 for Gr32a mutant males. n.s., p > 0.5, Mann-Whitney U test. (B) Total courtship indices of wild type males and Gr33a mutant males in courtship choice assays. Mean ± standard error of the mean (SEM), n = 21 for wild type males and 22 for Gr33a mutant males. n.s., p > 0.5, Mann-Whitney U test. (C) Total courtship indices of wild type males and Gr39a mutant males in courtship choice assays. Mean ± standard error of the mean (SEM), n = 20 for wild type males and 22 for Gr39a mutant males. Three asterisks, p < 0.001, Mann-Whitney U test. (D) Courtship indices of wild type males and Gr32a mutant males in courtship choice assays towards younger mates (bar colored white) and older ones (bar colored grey), respectively. Box-and-whisker plots for CIs show 1–99 percentiles and mean (+). Two asterisks, p < 0.01, three asterisks, p < 0.001, Related-samples Wilcoxon Signed Rank test. (E) Courtship indices of wild type males and Gr33a mutant males in courtship choice assays towards younger mates (white) and older ones (grey), respectively. Box-and-whisker plots for CIs show 1–99 percentiles and mean (+). n.s., p > 0.5, three asterisks, p < 0.001, Related-samples Wilcoxon Signed Rank test. (F) Preference indices of wild type males and Gr32a mutant males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.5, Mann-Whitney U test. (G) Preference indices of wild type males and Gr33a mutant males in courtship choice assays. Mean ± standard error of the mean (SEM). Three asterisks, p < 0.001, Mann-Whitney U test.

Since loss of Gr32a, Gr33a or Ppk29 does not affect males' sexual interest in females, we next examined their roles in males' preference behavior. In choice assays, Gr32aMI04430 males and Δppk29 males still prefer younger virgin females, while Gr33aGal4 males court both younger and older virgin females at the same intensity (Figure 1D, E and Figure S2A). To accurately quantify the extent of a male's preference for younger or older mates, we defined a preference index (PI), the relative difference between the courtship percentage towards younger females and that towards older ones in a choice assay: PI = [CIy − CIo]/[CIy + CIo]. PIs of Gr32aMI04430 and Δppk29 males are not substantially different from those of corresponding wild type controls, whereas PI of Gr33aGal4 males is completely abolished (Figure 1F, G and Figure S2B). These data indicate that malfunction of Gr33a eliminates males' preference for younger mates.

Gr33a is required for males' preference for younger mates

To further confirm the role of Gr33a in males' preference for younger mates, we examined another Gr33a mutant allele, Gr33a1, which deletes residues 1 to 199 of Gr33a13. Gr33a1 homozygous and trans-heterozygous (Gr33a1/Gr33aGal4) males present no preference behavior, yet the phenotype are fully rescued by the introduction of a UAS-Gr33a transgene13 in Gr33a1/Gr33aGal4 males (Gr33arescue) (Figure 2A and B). On the other hand, there is no difference among CIts of Gr33a mutant, Gr33arescue and wild type controls (Figure 2C), suggesting that Gr33a is not required for male-female courtship, but is necessary for males' preference for younger mates.

Figure 2. Gr33a modulates males' preference for younger mates.

Figure 2

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(A) Courtship indices of wild type males, Gr33aGal4/+ males, Gr33aGal4/Gr33a1 males, Gr33a1 males, and Gr33arescue males (Gr33aGal4/Gr33a1; UAS-Gr33a+/+) in courtship choice assays towards younger mates (white) and older ones (grey), respectively. Box-and-whisker plots for CIs show 1–99 percentiles and mean (+), n = 22 for wild type males, 23 for Gr33aGal4/+ males, 22 for Gr33aGal4/Gr33a1 males, 22 for Gr33a1 males, and 23 for Gr33arescue males. n.s., p > 0.05, three asterisks, p < 0.001, Related-samples Wilcoxon Signed Rank test. (B) Preference indices of wild type males, Gr33aGal4/+ males, Gr33aGal4/Gr33a1 males, Gr33a1 males, and Gr33arescue males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.05, three asterisks, p < 0.001, Kruskal-Wallis test, Dunn's post-hoc. (C) Total courtship indices of wild type males, Gr33aGal4/+ males, Gr33aGal4/Gr33a1 males, Gr33a1 males, and Gr33arescue males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.05, Kruskal-Wallis test, Dunn's post-hoc.

Activity of Gr33a neurons is essential for males' preference for younger mates

Gr33a is expressed widely in gustatory receptor neurons (GRNs) that are responsible for aversive chemicals13,21. To test whether the activity of Gr33a neurons is required for males' preference for younger mates, we inhibited synaptic transmissions in these neurons by expressing tetanus toxin light chain (TNT)22 under the control of Gr33aGal4 driver (Gr33aGal4/UAS-TNT). These TNT-expressing males display no preference for younger mates, whereas control males expressing an inactive version of the TNT transgene (TNTin)22 (Gr33aGal4/UAS-TNTin) exhibit a strong preference for younger mates (Figure 3A and B). On the other hand, males expressing TNT or TNTin display similar CIt (Figure 3C). These data demonstrate that the activity of Gr33a neurons is essential for males' preference for younger mates.

Figure 3. Activity of Gr33a neurons is essential for males' preference for younger mates.

Figure 3

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(A) Courtship indices of control males (Gr33aGal4/UAS-TNTin) and TNT expressing males (Gr33aGal4/UAS-TNT) in courtship choice assays towards younger mates (white) and older ones (grey), respectively. Box-and-whisker plots for CIs show 1-99 percentiles and mean (+), n = 23 for control males and 22 for TNT expressing males. n.s., p > 0.05, three asterisks, p < 0.001, Related-samples Wilcoxon Signed Rank test. (B) Preference indices of control males and TNT expressing males in courtship choice assays. Mean ± standard error of the mean (SEM). Three asterisks, p < 0.001, Mann-Whitney U test. (C) Total courtship indices of control males and TNT expressing males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.5, Mann-Whitney U test.

Expression of APP in Gr33a neurons results in dFoxO-dependent choice defect

Our previous findings suggest that pan-neuronal expression of APP, a potential causative protein of Alzheimer's disease (AD)23,24,25, abolishes males' preference for younger mates3. We asked whether expression of APP in Gr33a neurons (Gr33aGal4/UAS-APP) could interfere with their neuronal activity and affect males' preference behavior. We found that Gr33aGal4/UAS-APP males court both younger and older females at similar intensity (Figure 4A). The PI of Gr33aGal4/UAS-APP males is remarkably lower than that of control males (Gr33aGal4/+ and +/UAS-APP) (Figure 4B), whereas CIts among them remain unchanged (Figure 4C). Thus, expression of APP in Gr33a neurons is sufficient to block males' courtship preference for younger females. This perturbation is likely caused by a selective loss of Gr33a neurons, since ectopic expression of APP in Drosophila results in neuronal cell death26. Alternatively, overexpression of APP interferes with the expression of Gr33a or other critical signaling components, perhaps by causing ER stress. To address the issue, we examined Gr33a neuron in males' labella and foreleg tarsi, two sensory structures expressing Gr33a13, by the expression of GFP (Gr33aGal4; UAS-GFP), and found that overexpression of APP resulted in a significant loss of Gr33a neuron in labella and foreleg tarsi (Figure S3). Consistent with our recent study that the C-terminal APP intracellular domain (AICD) is required for APP to trigger cell death27, males overexpressing in Gr33a neurons a truncated APP that lacks AICD (APPΔCT) exhibit similar PI and CI to those of control males (Figure 4A, B and C).

Figure 4. APP expression in Gr33a neurons abolishes males' preference.

Figure 4

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(A) Courtship indices of Gr33aGal4/+ and +/UAS-APP control males, Gr33aGal4/UAS-APP males, Gr33aGal4/UAS-APPΔCT males, Gr33aGal4/+; dfoxOΔ94/+ males and Gr33aGal4/UAS-APP; dfoxOΔ94/+ males in courtship choice assays towards younger mates (white) and older ones (grey), respectively. Box-and-whisker plots for CIs show 1–99 percentiles and mean (+), n = 22 for control males, 21 for +/UAS-APP males, 23 for Gr33aGal4/UAS-APP males, 25 for Gr33aGal4/UAS-APPΔCT males, 21 for Gr33aGal4/+; dfoxOΔ94/+ males and 21 for Gr33aGal4/UAS-APP; dfoxOΔ94/+ males. n.s., p > 0.05, two asterisks, p < 0.01, three asterisks, p < 0.001, Related-samples Wilcoxon Signed Rank test. (B) Preference indices of Gr33aGal4/+ and +/UAS-APP control males, Gr33aGal4/UAS-APP males, Gr33aGal4/UAS-APPΔCT males, Gr33aGal4/+; dfoxOΔ94/+ males and Gr33aGal4/UAS-APP; dfoxOΔ94/+ males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.05, three asterisks, p < 0.001, Kruskal-Wallis test, Dunn's post-hoc. (C) Total courtship indices of Gr33aGal4/+ and +/UAS-APP control males, Gr33aGal4/UAS-APP males, Gr33aGal4/UAS-APPΔCT males, Gr33aGal4/+; dfoxOΔ94/+ males and Gr33aGal4/UAS-APP; dfoxOΔ94/+ males in courtship choice assays. Mean ± standard error of the mean (SEM). n.s., p > 0.05, Kruskal-Wallis test, Dunn's post-hoc.

The forkhead box O (FoxO) transcription factors have been highly conserved during evolution, and are postulated to modulate various physiological processes, including cell proliferation and differentiation, apoptosis and metabolism28,29,30,31. Recently, we identified dFoxO as a crucial downstream factor that mediates APP-induced cell death and locomotion defect in Drosophila27. To examine whether dFoxO is also involved in APP-triggered male courtship preference defect, we introduced a dfoxO null mutation (dfoxOΔ94)32,33 into males expressing APP in Gr33a neurons. We found that deleting one copy of endogenous dfoxo gene significantly rescues the preference defect of Gr33aGal4/UAS-APP males (Figure 4A and B), while the CIts show no discrepancy among all groups (Figure 4C). These results, taken together, manifest that ectopic expression of APP in Gr33a neurons disrupts the activity of these neurons and acutely dispels males' preference for younger mates. Such function of APP in choice behavior, which is mediated by dFoxO, could be employed as a potential AD model for cognitive study.

Female CHC profiles change with age

Cuticular hydrocarbons (CHCs) are reported to serve as sex pheromones which act through gustatory receptors to modulate male courtship behavior7. Among all the CHCs produced by Drosophila melanogaster, 7-tricosene (7-T) (C23) is repulsive to males; 7-pentacosene (7-P) (C25) has complex roles in courtship whereas 7,11-heptacosadiene (7,11-HD) (C27) and 7,11-nonacosadiene (7,11-ND) (C29) are attractive to males4. To explore the difference in CHC profiles between younger and older virgin females, we performed gas chromatography and mass spectrometry. We found that the concentrations of most female specific dienes and alkenes were lower on younger virgin females than on older ones (Figure 5 and Table S1). Thus, we conclude that female CHC profiles change with age, with the major female specific CHCs increase by aging.

Figure 5. CHC Profiles of younger and older virgin females.

Figure 5

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(A) and (A′) The chromatogram plots the area of each peak associated with a compound in units of abundance with column retention time on the x-axis. (A) shows the profile of younger virgin females and (A′) shows that of older ones. (B) Relative abundance (injection standard nC26 is 100) of candidate dienes and alkenes on virgin females. Bar colored white indicates younger virgin females and bar colored grey indicates older ones. n = 4–5 for each compound. n.s., p > 0.05, asterisk, p < 0.05, two asterisks, p < 0.01, Mann-Whitney U test.

Discussion

The function of Grs in males' preference behavior

Drosophila male courtship choice has been frequently applied for studying decision making in animals34, yet most of the past studies have focused on male courtship choices between likes and dislikes, such as court towards females vs. males35, or virgin vs. non-virgin females36,37. We have previously characterized a choice behavior between two equally-liked options: mature virgin females, whether younger or older, were similarly attractive to naive males; nevertheless, when given the option, males turn out to be picky and prefer younger virgin females to older ones3. Here we found that a gustatory receptor, Gr33a, is necessary for males' preference for younger mates. Gr33a is thought to be necessary to inhibit homosexual behavior13,21; its role in heterosexual behavior, however, is rarely pondered. In this study, we reveal the critical role of Gr33a in males' preference for younger mates. Furthermore, ectopic expression of APP in Gr33a neurons eliminates males' preference behavior, and such function is mediated by dFoxO, a recently reported downstream factor of APP27. Therefore, our work demonstrates the genetic interaction of APP and dFoxO in Gr33a neurons, which modulates males' preference for younger mates.

APP is identified as a potential causative protein of AD, a common progressive neurodegenerative disorder25, in which cognitive decline is the prime symptom23,24,38. Although Drosophila has long been utilized for building AD models to investigate the pathogenesis and possible cure for AD39,40, accepted Drosophila AD models are limited to locomotion model and life span model41,42,43, which have little correlation with cognitive ability. Our findings, however, have offered the possibility for establishing a novel Drosophila AD model that is related to cognitive ability.

The role of CHCs in males' preference

In all CHCs produced by files, 7, 11-HD and 7, 11-ND have been identified as female specific aphrodisiac pheromones to Drosophila melanogaster males20. Our GC and MS results suggest that both 7, 11-HD and 7, 11-ND are expressed at lower concentration in younger virgin females than the older ones. Hence, it appears unlikely that 7, 11-HD or 7, 11-ND is the cause that leads males to court younger virgin females more vigorously than the older ones. On the contrary, since Gr33a has been reported as a receptor of aversive odors13,21, it is more likely that older females produce certain aversive odors that can be recognized by males and repel them. Consistent with this explanation, we found that the concentrations of most detected CHCs are significantly higher on the older virgin females than the younger ones. Nevertheless, at this stage we are unable to identify the CHC(s) that serves as the aversive pheromone to males. Besides, we cannot exclude the possibility that younger virgin females produce unknown attractive pheromones other than 7, 11-HD or 7, 11-ND. However, all CHCs we have detected on younger virgin females also present on older ones at a similar or higher lever. Thus, we draw the tentative conclusion that younger virgin females do not produce more attractive pheromones than the older ones. Our results, taken together, unravel the role of bitter sensory Gr33a neurons in males' preference for younger mates and infer that older females might produce certain aversive odors that cause males to turn to younger mates.

Supplementary Material

Supplementary Information

Supplementary information

srep07777-s1.pdf (501.1KB, pdf)

Acknowledgments

The authors thank Dr. Zuoren Wang, Dr. Kristin Scott, Dr. Aike Guo and Dr. Linda Partridge, the Bloomington stock center for fly stocks, Dr. Margaret Ho and members of Xue lab for discussion and comments. This work is supported by the National Basic Research Program of China (973 Program) (2011CB943903), National Natural Science Foundation of China (31071294, 31171413, 31371490), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20120072110023), and Shanghai Committee of Science and Technology (09DZ2260100 and 14JC1406000).

Footnotes

Author Contributions Y.H. and L.X. conceived the project and designed the experiments; Y.H., Y.H. and Y.S. performed the experiments; X.W., Y.M. and E.L. contributed unpublished reagents; Y.H. and Y.H. analyzed the data; Y.H. and L.X. wrote the manuscript.

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