Neurobiology of Aging xxx (2014) 1e5
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Aging-related neurodegeneration eliminates male courtship choice in Drosophila Yujia Hu, Yi Han, Xingjun Wang, Lei Xue* Department of Interventional Radiology, Shanghai 10th People’s Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2013 Received in revised form 1 February 2014 Accepted 27 February 2014
Choices between 2 options, one liked and one disliked, are effortless for an animal, whereas those among 2 equally liked options are more difficult to determine and might depend on an unknown mechanism. Here, we report that in the fruit fly Drosophila melanogaster, both younger virgin females and older ones are “liked options” to males. However, when given the choice, males tend to be fastidious and prefer younger virgin females to older ones. Besides, aging eliminates males’ preference for younger mates, which can be mimicked by ectopically expressing the human amyloid precursor protein in their central nervous system. Furthermore, we examined the effect of neurodegeneration in Drosophila courtship circuit and confirmed that male courtship preference for younger mates was abrogated by neurodegeneration. Our work, thus characterizes a novel choice behavior that can be decisions after comparison and also reveals the critical role of neurodegeneration in this behavior, which provides new insights on decision-making mechanisms. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Aging Neurodegeneration Male courtship Drosophila
1. Introduction
2. Methods
In the fruit fly Drosophila melanogaster, male courtship behavior is used as a paradigm for decision making in animals. Most of past studies on decision making focused on male courtship choices between likes and dislikes, such as court toward females versus males (Demir and Dickson, 2005; Thistle et al., 2012), or virgin versus nonvirgin females (Keleman et al., 2012; Siegel and Hall, 1979). Nevertheless, how males choose their courting subjects from numerous attractive virgin females remains largely unknown. To observe Drosophila males’ courting choice we conducted a series of single-pair assays and choice-assays by paring males with females of different ages. To check the role of aging and neurodegeneration in males’ choice behavior, we examined wild-type males of different ages and males ectopically expressing neurodegeneration candidate proteins in the nervous system. We found that Drosophila males prefer younger females to older ones and that this preference declines with age and are eliminated by neuronal specific expression of neurodegeneration candidate proteins.
2.1. Drosophila strains
* Corresponding author at: Department of Interventional Radiology, Shanghai 10th People’s Hospital, Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, 1239 Siping Road, Shanghai, China. Tel./fax: þ86 21 65985407. E-mail address: [email protected] (L. Xue). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.02.026
Oregon R, w1118, elav-Gal4, UAS-LacZ, and UAS-APP were obtained from Bloomington Drosophila Stock Center. Fruitless-Gal4 was a gift from Dr Zuoren Wang and UAS-Httex1-Q72-eGFP was generously provided by Dr Sheng Zhang. 2.2. Fly rearing Drosophila stocks were maintained on a standard corn flour, yeast, and agar medium under a 12-hour light and 12-hour dark cycle at 25 C. Naive male and virgin female flies were collected at eclosion. Males were housed individually and females in groups of 10 in vials and transferred to new vials containing fresh medium every 3 days by using light CO2. 2.3. Courtship behavior assays Courtship behavior assays were performed at approximately the same time each day (within 1 hour at the beginning of the 12-hour illumination half of the cycle) (Grosjean et al., 2011) in round observation chambers (1.5 cm in diameter and 0.3 cm deep) by observing male courtship behavior at 25 C. Single-pair assays were
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performed by pairing a naive male with either a younger wild-type virgin female or an older one, and choice-assays by pairing a naive male together with 2 younger wild type virgin females and 2 older ones. To eliminate the individual variances (e.g., slight difference of body sizes) we used 4 females (2 younger and 2 older) while in the control experiments, which determined eye color effect we used 4 females of the same age. All tests were recorded for 10 minutes 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 results (Moehring and Mackay, 2004), tests in which male mated successfully within 10 minutes were excluded before analysis. The courtship index (CI) was calculated as the percentage of time that a male courted the females during a 10-minute period. 2.4. Statistical analysis Because CIs were generally not normally distributed, statistical comparisons of intragroup CIs (CIy and CIo) in choice-assays used related-samples Wilcoxon signed-rank test and intergroup CIs in single-pair assays were compared using ManneWhitney U test. Besides, total courtship index (CIt) and preference index (PI) in choice-assays were compared using KruskaleWallis test followed by the post hoc Dunn test. 3. Results and discussion We counted 3-day-old virgin females as younger females and 30-day-old virgin females as older ones. A male’s courtship
behavior was measured by a CI. In single-pair assays, 3-day-old naive wild type (Oregon R) males courted intensively the younger wild type (Oregon R) females or older ones in similar strength (Fig. 1A), indicating that mature virgin females, whether younger or older, were both attractive to naive males. In the subsequent choice-assays, eye colors (Oregon R as red and w1118 as white) were used as marks to distinguish females of different ages: younger and older females were marked as red and white or vice versa. To exclude the possibility that females’ eye colors would affect males’ courtship choice, we conducted an eye color control choice-assay in which naive wild-type males were provided with virgin females of same age (3 days) but different eye colors (red or white). We found that males exhibited no preference for the eye color of their mates (Fig. 1B). When females of different ages were provided in the choice-assays, however, males courted younger females more vigorously than older ones, regardless of their eye colors (Fig. 1C). These results suggest that Drosophila males’ preference behavior for younger mates is not simply an instinctive impulse but rather a decision after comparison. During the process of development, discriminating ability declines as an outcome of aging (Germine et al., 2011). To examine whether the preference for younger mates in Drosophila males is age-dependent, we introduced 30-day-old and 60-day-old naive wild type (Oregon R) males into the choice-assays. To accurately quantify the extent of a male’s preference for younger or older females, we defined a PI, the relative difference between his courtship percentage toward younger females and that toward older ones in a choice assay: PI ¼ (CIy CIo)/(CIy þ CIo). Besides, to assess a male’s courtship ability we defined a CIt, the percentage of his total courtship time in a choice assay: CIt ¼ (CIy þ CIo). Compared with 3-day-old males, the preference for younger females was diminished in 30-day-old males and abolished in 60-day-old males (Fig. 2A and B). On the other hand, the male’s
Fig. 1. Drosophila male courtship preference for younger mates is a decision after comparison. (A) Courtship indices of 3-day-old naive wild type (Oregon R) males toward younger virgin wild-type females (bar colored white) or older ones (gray) in single-pair assays (labeled as WT [3D]S). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 26 paired with younger females, 31 with older ones; n.s., p > 0.5 between intergroup courtships toward younger females or older ones (ManneWhitney U test). (B) Courtship indices of 3-day-old naive wild type (Oregon R) males toward 3-day-old red eye virgin wild type (Oregon R) females (bar colored red) and 3-day-old white eye virgin (w1118) females (white) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24; n.s., p > 0.5 between intragroup courtships toward red eye females and white eye ones (related-samples Wilcoxon signed-rank test). (C) Courtship indices of 3-day-old naive wild type (Oregon R) males toward younger virgin wild-type females (white) or older ones (gray) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24. Three asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). Abbreviation: CIs, courtship indices.
Y. Hu et al. / Neurobiology of Aging xxx (2014) 1e5
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Fig. 2. Aging eliminates wild type Drosophila male courtship preference for younger mates. (A) Courtship indices of 3, 30 and 60-day-old naive wild type males (Oregon R) in choiceassays toward younger virgin wild-type females (white) and older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24 for 3-day-old males, 22 for 30-day-old males and 30 for 60-day-old males; n.s., p > 0.5, 2 asterisks, p < 0.01, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (relatedsamples Wilcoxon signed-rank test). (B) Preference indices of 3, 30, and 60-day-old naive wild-type males (Oregon R) in choice-assays. Mean standard error of the mean (SEM), n ¼ 24 for 3-day-old males, 22 for 30-day-old males, and 30 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3, 30, and 60-day-old naive wild type males (Oregon R) in choice-assays. Mean standard error of the mean (SEM), n ¼ 24 for 3-day-old males, 22 for 30-dayold males, and 30 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (D) Courtship indices of 3, 30, and 60-day-old naive wild type (Oregon R) males toward 3-day-old red eye virgin wild type (Oregon R) females (bar colored red) and 3-day-old white eye virgin (w1118) females (white) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24 for 3-day-old males, 20 for 30-day-old males, and 22 for 60-day-old males; n.s., p > 0.5 between intragroup courtships toward red eye females and white eye ones (related-samples Wilcoxon signed-rank test). Abbreviation: CIs, courtship indices.
Fig. 3. Aging eliminates w1118 Drosophila male courtship preference for younger mates. (A) Courtship indices of 3, 30, and 60-day-old naive w1118 males in choice-assays toward younger virgin wild-type females (white) and older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 14 for 3-day-old males,16 for 30-day-old males, and 16 for 60 day-old males; n.s., p > 0.5, 2 asterisks, p < 0.01, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3, 30, and 60-day-old naive wild type males (w1118) in choice-assays. Mean standard error of the mean (SEM), n ¼ 14 for 3-day-old males,16 for 30-day-old males, and 16 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3, 30, and 60-day-old naive wild type males (w1118) in choice-assays. Mean standard error of the mean (SEM), n ¼ 14 for 3-day-old males, 16 for 30-day-old males, and 16 for 60-day-old males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). Abbreviation: CIs, courtship indices.
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Fig. 4. Expression of APP in Drosophila males’ CNS can cause loss of their preference for younger mates. (A) Courtship indices of 3-day-old naive control males (elav-GAL4), LacZexpressing males (elav > LacZ), UAS-APP males and APP-expressing males (elav > APP) in choice-assays toward younger females (white) or older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 28 for control males, 26 for LacZ-expressing males, 25 for UAS-APP males and 33 for APP-expressing males; n.s., p > 0.5, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, and APP-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 26 for LacZexpressing males, 25 for UAS-APP males, and 33 for APP-expressing males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with control males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, and APP-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 26 for LacZ-expressing males, 25 for UAS-APP males, and 33 for APP-expressing males; n.s., p > 0.5 compared with control males (KruskaleWallis test, Dunn post hoc). Abbreviations: APP, amyloid precursor protein; CIs, courtship indices; CNS, central nervous system.
overall courtship activity was only slightly decreased in aged males, as compared with that of 3-day-old males (Fig. 2C). To examine whether aged males develop a particular preference for females with a certain eye color, we performed eye color control choice-assay in which naive-wild type males with increasing age (3-day-old, 30-day-old, and 60-day-old, respectively) were paired with virgin females of same age (3-day-old) but different eye colors (red or white). Results showed aged males courted red or white eye females of the same age at similar intensity, suggesting female’s eye colors do not affect male’s courtship choice (Fig. 2D). The fact that aged males displayed significantly reduced PI (Fig. 2B) documents that aging eliminates males’ ability of discriminating younger females from older ones. We also tested this preference behavior in another Drosophila strain (w1118). Similar to Oregon R males, the naive w1118 males prefer younger mates whereas such preference diminishes with aging (Fig. 3), indicating that loss of
males’ preference for younger mates is a general phenomenon of aging in D. melanogaster. Aging can cause diverse array of physiological effects, such as impairment of motility, a decrease in sexual desire and loss of memory, etc. (Iliadi and Boulianne, 2010; Wang et al., 2011). However, cognitive decline associated with aging is closely related to neurodegeneration resulted from overexpression of human amyloid precursor protein (APP) (Greeve et al., 2004; Salehi et al., 2006; Zhao et al., 2010). APP is identified as a potential causative protein of Alzheimer’s disease, which is a common progressive neurodegenerative disorder (Lovestone, 1995). To investigate the role of neurodegeneration in the males’ preference behavior we expressed full length human APP in Drosophila under the panneuronal elav-Gal4 driver (Koushika et al., 1996). We introduced 3-day-old naive APP-expressing males as courters into choiceassays. APP-expressing males were unable to distinguish younger
Fig. 5. Drosophila male courtship preference for younger mates was abrogated by neurodegeneration. (A) Courtship indices of 3-day-old naive control males (fruitless-GAL4), LacZexpressing males (fruitless > LacZ), UAS-APP males, APP-expressing males (fruitless > APP), UAS-Htt males and Htt-expressing males (fruitless > Htt) in choice-assays toward younger females (white) or older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UASAPP males, 24 for APP-expressing males, 23 for UAS-Htt males, and 28 for Htt-expressing males; n.s., p > 0.5, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, APP-expressing males, UAS-Htt males, and Htt-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UAS-APP males, 24 for APP-expressing males, 23 for UAS-Htt males, and 28 for Htt-expressing males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with control males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, APP-expressing males, UAS-Htt males, and Htt-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UAS-APP males, 24 for APP-expressing males, 23 for UASHtt males, and 28 for Htt-expressing males; n.s., p > 0.5 compared with control males (KruskaleWallis test, Dunn post hoc). Abbreviation: APP, amyloid precursor protein.
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females from older ones (Fig. 4A and B), resemble that of 60-dayold males (Figs. 2 and 3). In contrast, APP-expressing males showed no defects in CIt compared with control ones (Fig. 4C), indicating that APP expression does not compromise males’ overall courtship ability or courting interest in females. The PI of APPexpressing males decreased significantly compared with that of the controls (Fig. 4B), suggesting male’s discriminating ability was abrogated by pan-neuronal expression of APP. To test the specific effect of neurodegeneration in Drosophila courtship circuit, we used the courtship neuron specific fruitless-Gal4 (Demir and Dickson, 2005) to drive the expression of APP or a mutated form of huntingtin with a polyglutamine expansion (Httex1-Q72; Zhang et al., 2010) that is responsible for the Huntington disease (Nasir et al., 1995; Romero et al., 2008; Steffan et al., 2004). Expression of APP or Httex1-Q72 driven by fruitless-Gal4 abolished males’ courtship preference for younger mates (Fig. 5A and B) but did not affect their total courtship index (CIt) (Fig. 5C). Taken together, these results imply that Drosophila males’ courtship preference for younger females, as compared with their courtship ability and interest in females, is an advanced discriminating ability that could be eliminated by neurodegeneration. Although the courtship ritual itself does not cost too much (Dickson, 2008), males still try their best to obtain the maximum benefit of reproductive investment, by nature or from learning. Adult males are typically repulsive to other males hence futile homosexual behavior can be inherently avoided in wild-type Drosophila males (Stockinger et al., 2005). While mature females are usually attractive to naive males even if the females have recently mated (Siegel and Hall, 1979), prior experience of rejection from unreceptive mated females renders males more sensitive to cis-vaccenyl acetate (cVA), an aversive pheromone which can be transferred from males to females during mating (Keleman et al., 2012) and teaches them to avoid meaningless courtship toward mated females. These choices, more or less, are between likes and dislikes. The males’ preference behavior we described here, however, is a choice between liked options. We anticipate our findings to be a starting point for delving into neural circuits that govern choosing behavior after prudent comparison rather than intrinsic impulse. Disclosure statement The authors have no conflicts of interest to declare. Acknowledgements The authors thank Drs Zuoren Wang and Sheng Zhang, the Bloomington stock center for fly stocks, Beika Lu, Edward Yao, 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) (2010CB944901, 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).
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Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Aging-related neurodegeneration eliminates male courtship choice in Drosophila Yujia Hu, Yi Han, Xingjun Wang, Lei Xue* Department of Interventional Radiology, Shanghai 10th People’s Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2013 Received in revised form 1 February 2014 Accepted 27 February 2014
Choices between 2 options, one liked and one disliked, are effortless for an animal, whereas those among 2 equally liked options are more difficult to determine and might depend on an unknown mechanism. Here, we report that in the fruit fly Drosophila melanogaster, both younger virgin females and older ones are “liked options” to males. However, when given the choice, males tend to be fastidious and prefer younger virgin females to older ones. Besides, aging eliminates males’ preference for younger mates, which can be mimicked by ectopically expressing the human amyloid precursor protein in their central nervous system. Furthermore, we examined the effect of neurodegeneration in Drosophila courtship circuit and confirmed that male courtship preference for younger mates was abrogated by neurodegeneration. Our work, thus characterizes a novel choice behavior that can be decisions after comparison and also reveals the critical role of neurodegeneration in this behavior, which provides new insights on decision-making mechanisms. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Aging Neurodegeneration Male courtship Drosophila
1. Introduction
2. Methods
In the fruit fly Drosophila melanogaster, male courtship behavior is used as a paradigm for decision making in animals. Most of past studies on decision making focused on male courtship choices between likes and dislikes, such as court toward females versus males (Demir and Dickson, 2005; Thistle et al., 2012), or virgin versus nonvirgin females (Keleman et al., 2012; Siegel and Hall, 1979). Nevertheless, how males choose their courting subjects from numerous attractive virgin females remains largely unknown. To observe Drosophila males’ courting choice we conducted a series of single-pair assays and choice-assays by paring males with females of different ages. To check the role of aging and neurodegeneration in males’ choice behavior, we examined wild-type males of different ages and males ectopically expressing neurodegeneration candidate proteins in the nervous system. We found that Drosophila males prefer younger females to older ones and that this preference declines with age and are eliminated by neuronal specific expression of neurodegeneration candidate proteins.
2.1. Drosophila strains
* Corresponding author at: Department of Interventional Radiology, Shanghai 10th People’s Hospital, Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, 1239 Siping Road, Shanghai, China. Tel./fax: þ86 21 65985407. E-mail address: [email protected] (L. Xue). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.02.026
Oregon R, w1118, elav-Gal4, UAS-LacZ, and UAS-APP were obtained from Bloomington Drosophila Stock Center. Fruitless-Gal4 was a gift from Dr Zuoren Wang and UAS-Httex1-Q72-eGFP was generously provided by Dr Sheng Zhang. 2.2. Fly rearing Drosophila stocks were maintained on a standard corn flour, yeast, and agar medium under a 12-hour light and 12-hour dark cycle at 25 C. Naive male and virgin female flies were collected at eclosion. Males were housed individually and females in groups of 10 in vials and transferred to new vials containing fresh medium every 3 days by using light CO2. 2.3. Courtship behavior assays Courtship behavior assays were performed at approximately the same time each day (within 1 hour at the beginning of the 12-hour illumination half of the cycle) (Grosjean et al., 2011) in round observation chambers (1.5 cm in diameter and 0.3 cm deep) by observing male courtship behavior at 25 C. Single-pair assays were
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performed by pairing a naive male with either a younger wild-type virgin female or an older one, and choice-assays by pairing a naive male together with 2 younger wild type virgin females and 2 older ones. To eliminate the individual variances (e.g., slight difference of body sizes) we used 4 females (2 younger and 2 older) while in the control experiments, which determined eye color effect we used 4 females of the same age. All tests were recorded for 10 minutes 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 results (Moehring and Mackay, 2004), tests in which male mated successfully within 10 minutes were excluded before analysis. The courtship index (CI) was calculated as the percentage of time that a male courted the females during a 10-minute period. 2.4. Statistical analysis Because CIs were generally not normally distributed, statistical comparisons of intragroup CIs (CIy and CIo) in choice-assays used related-samples Wilcoxon signed-rank test and intergroup CIs in single-pair assays were compared using ManneWhitney U test. Besides, total courtship index (CIt) and preference index (PI) in choice-assays were compared using KruskaleWallis test followed by the post hoc Dunn test. 3. Results and discussion We counted 3-day-old virgin females as younger females and 30-day-old virgin females as older ones. A male’s courtship
behavior was measured by a CI. In single-pair assays, 3-day-old naive wild type (Oregon R) males courted intensively the younger wild type (Oregon R) females or older ones in similar strength (Fig. 1A), indicating that mature virgin females, whether younger or older, were both attractive to naive males. In the subsequent choice-assays, eye colors (Oregon R as red and w1118 as white) were used as marks to distinguish females of different ages: younger and older females were marked as red and white or vice versa. To exclude the possibility that females’ eye colors would affect males’ courtship choice, we conducted an eye color control choice-assay in which naive wild-type males were provided with virgin females of same age (3 days) but different eye colors (red or white). We found that males exhibited no preference for the eye color of their mates (Fig. 1B). When females of different ages were provided in the choice-assays, however, males courted younger females more vigorously than older ones, regardless of their eye colors (Fig. 1C). These results suggest that Drosophila males’ preference behavior for younger mates is not simply an instinctive impulse but rather a decision after comparison. During the process of development, discriminating ability declines as an outcome of aging (Germine et al., 2011). To examine whether the preference for younger mates in Drosophila males is age-dependent, we introduced 30-day-old and 60-day-old naive wild type (Oregon R) males into the choice-assays. To accurately quantify the extent of a male’s preference for younger or older females, we defined a PI, the relative difference between his courtship percentage toward younger females and that toward older ones in a choice assay: PI ¼ (CIy CIo)/(CIy þ CIo). Besides, to assess a male’s courtship ability we defined a CIt, the percentage of his total courtship time in a choice assay: CIt ¼ (CIy þ CIo). Compared with 3-day-old males, the preference for younger females was diminished in 30-day-old males and abolished in 60-day-old males (Fig. 2A and B). On the other hand, the male’s
Fig. 1. Drosophila male courtship preference for younger mates is a decision after comparison. (A) Courtship indices of 3-day-old naive wild type (Oregon R) males toward younger virgin wild-type females (bar colored white) or older ones (gray) in single-pair assays (labeled as WT [3D]S). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 26 paired with younger females, 31 with older ones; n.s., p > 0.5 between intergroup courtships toward younger females or older ones (ManneWhitney U test). (B) Courtship indices of 3-day-old naive wild type (Oregon R) males toward 3-day-old red eye virgin wild type (Oregon R) females (bar colored red) and 3-day-old white eye virgin (w1118) females (white) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24; n.s., p > 0.5 between intragroup courtships toward red eye females and white eye ones (related-samples Wilcoxon signed-rank test). (C) Courtship indices of 3-day-old naive wild type (Oregon R) males toward younger virgin wild-type females (white) or older ones (gray) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24. Three asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). Abbreviation: CIs, courtship indices.
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Fig. 2. Aging eliminates wild type Drosophila male courtship preference for younger mates. (A) Courtship indices of 3, 30 and 60-day-old naive wild type males (Oregon R) in choiceassays toward younger virgin wild-type females (white) and older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24 for 3-day-old males, 22 for 30-day-old males and 30 for 60-day-old males; n.s., p > 0.5, 2 asterisks, p < 0.01, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (relatedsamples Wilcoxon signed-rank test). (B) Preference indices of 3, 30, and 60-day-old naive wild-type males (Oregon R) in choice-assays. Mean standard error of the mean (SEM), n ¼ 24 for 3-day-old males, 22 for 30-day-old males, and 30 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3, 30, and 60-day-old naive wild type males (Oregon R) in choice-assays. Mean standard error of the mean (SEM), n ¼ 24 for 3-day-old males, 22 for 30-dayold males, and 30 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (D) Courtship indices of 3, 30, and 60-day-old naive wild type (Oregon R) males toward 3-day-old red eye virgin wild type (Oregon R) females (bar colored red) and 3-day-old white eye virgin (w1118) females (white) in choice-assays. Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 24 for 3-day-old males, 20 for 30-day-old males, and 22 for 60-day-old males; n.s., p > 0.5 between intragroup courtships toward red eye females and white eye ones (related-samples Wilcoxon signed-rank test). Abbreviation: CIs, courtship indices.
Fig. 3. Aging eliminates w1118 Drosophila male courtship preference for younger mates. (A) Courtship indices of 3, 30, and 60-day-old naive w1118 males in choice-assays toward younger virgin wild-type females (white) and older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 14 for 3-day-old males,16 for 30-day-old males, and 16 for 60 day-old males; n.s., p > 0.5, 2 asterisks, p < 0.01, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3, 30, and 60-day-old naive wild type males (w1118) in choice-assays. Mean standard error of the mean (SEM), n ¼ 14 for 3-day-old males,16 for 30-day-old males, and 16 for 60-day-old males. Asterisks, p < 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3, 30, and 60-day-old naive wild type males (w1118) in choice-assays. Mean standard error of the mean (SEM), n ¼ 14 for 3-day-old males, 16 for 30-day-old males, and 16 for 60-day-old males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with 3-day-old males (KruskaleWallis test, Dunn post hoc). Abbreviation: CIs, courtship indices.
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Fig. 4. Expression of APP in Drosophila males’ CNS can cause loss of their preference for younger mates. (A) Courtship indices of 3-day-old naive control males (elav-GAL4), LacZexpressing males (elav > LacZ), UAS-APP males and APP-expressing males (elav > APP) in choice-assays toward younger females (white) or older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 28 for control males, 26 for LacZ-expressing males, 25 for UAS-APP males and 33 for APP-expressing males; n.s., p > 0.5, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, and APP-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 26 for LacZexpressing males, 25 for UAS-APP males, and 33 for APP-expressing males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with control males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, and APP-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 26 for LacZ-expressing males, 25 for UAS-APP males, and 33 for APP-expressing males; n.s., p > 0.5 compared with control males (KruskaleWallis test, Dunn post hoc). Abbreviations: APP, amyloid precursor protein; CIs, courtship indices; CNS, central nervous system.
overall courtship activity was only slightly decreased in aged males, as compared with that of 3-day-old males (Fig. 2C). To examine whether aged males develop a particular preference for females with a certain eye color, we performed eye color control choice-assay in which naive-wild type males with increasing age (3-day-old, 30-day-old, and 60-day-old, respectively) were paired with virgin females of same age (3-day-old) but different eye colors (red or white). Results showed aged males courted red or white eye females of the same age at similar intensity, suggesting female’s eye colors do not affect male’s courtship choice (Fig. 2D). The fact that aged males displayed significantly reduced PI (Fig. 2B) documents that aging eliminates males’ ability of discriminating younger females from older ones. We also tested this preference behavior in another Drosophila strain (w1118). Similar to Oregon R males, the naive w1118 males prefer younger mates whereas such preference diminishes with aging (Fig. 3), indicating that loss of
males’ preference for younger mates is a general phenomenon of aging in D. melanogaster. Aging can cause diverse array of physiological effects, such as impairment of motility, a decrease in sexual desire and loss of memory, etc. (Iliadi and Boulianne, 2010; Wang et al., 2011). However, cognitive decline associated with aging is closely related to neurodegeneration resulted from overexpression of human amyloid precursor protein (APP) (Greeve et al., 2004; Salehi et al., 2006; Zhao et al., 2010). APP is identified as a potential causative protein of Alzheimer’s disease, which is a common progressive neurodegenerative disorder (Lovestone, 1995). To investigate the role of neurodegeneration in the males’ preference behavior we expressed full length human APP in Drosophila under the panneuronal elav-Gal4 driver (Koushika et al., 1996). We introduced 3-day-old naive APP-expressing males as courters into choiceassays. APP-expressing males were unable to distinguish younger
Fig. 5. Drosophila male courtship preference for younger mates was abrogated by neurodegeneration. (A) Courtship indices of 3-day-old naive control males (fruitless-GAL4), LacZexpressing males (fruitless > LacZ), UAS-APP males, APP-expressing males (fruitless > APP), UAS-Htt males and Htt-expressing males (fruitless > Htt) in choice-assays toward younger females (white) or older ones (gray). Box-and-whisker plots for CIs show 1e99 percentiles and mean (þ), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UASAPP males, 24 for APP-expressing males, 23 for UAS-Htt males, and 28 for Htt-expressing males; n.s., p > 0.5, 3 asterisks, p < 0.001 between intragroup courtships toward younger females and older ones (related-samples Wilcoxon signed-rank test). (B) Preference indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, APP-expressing males, UAS-Htt males, and Htt-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UAS-APP males, 24 for APP-expressing males, 23 for UAS-Htt males, and 28 for Htt-expressing males; n.s., p > 0.05, 3 asterisks, p < 0.001 compared with control males (KruskaleWallis test, Dunn post hoc). (C) Total courtship indices of 3-day-old naive control males, LacZ-expressing males, UAS-APP males, APP-expressing males, UAS-Htt males, and Htt-expressing males in choice-assays. Mean standard error of the mean (SEM), n ¼ 28 for control males, 23 for LacZ-expressing males, 25 for UAS-APP males, 24 for APP-expressing males, 23 for UASHtt males, and 28 for Htt-expressing males; n.s., p > 0.5 compared with control males (KruskaleWallis test, Dunn post hoc). Abbreviation: APP, amyloid precursor protein.
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females from older ones (Fig. 4A and B), resemble that of 60-dayold males (Figs. 2 and 3). In contrast, APP-expressing males showed no defects in CIt compared with control ones (Fig. 4C), indicating that APP expression does not compromise males’ overall courtship ability or courting interest in females. The PI of APPexpressing males decreased significantly compared with that of the controls (Fig. 4B), suggesting male’s discriminating ability was abrogated by pan-neuronal expression of APP. To test the specific effect of neurodegeneration in Drosophila courtship circuit, we used the courtship neuron specific fruitless-Gal4 (Demir and Dickson, 2005) to drive the expression of APP or a mutated form of huntingtin with a polyglutamine expansion (Httex1-Q72; Zhang et al., 2010) that is responsible for the Huntington disease (Nasir et al., 1995; Romero et al., 2008; Steffan et al., 2004). Expression of APP or Httex1-Q72 driven by fruitless-Gal4 abolished males’ courtship preference for younger mates (Fig. 5A and B) but did not affect their total courtship index (CIt) (Fig. 5C). Taken together, these results imply that Drosophila males’ courtship preference for younger females, as compared with their courtship ability and interest in females, is an advanced discriminating ability that could be eliminated by neurodegeneration. Although the courtship ritual itself does not cost too much (Dickson, 2008), males still try their best to obtain the maximum benefit of reproductive investment, by nature or from learning. Adult males are typically repulsive to other males hence futile homosexual behavior can be inherently avoided in wild-type Drosophila males (Stockinger et al., 2005). While mature females are usually attractive to naive males even if the females have recently mated (Siegel and Hall, 1979), prior experience of rejection from unreceptive mated females renders males more sensitive to cis-vaccenyl acetate (cVA), an aversive pheromone which can be transferred from males to females during mating (Keleman et al., 2012) and teaches them to avoid meaningless courtship toward mated females. These choices, more or less, are between likes and dislikes. The males’ preference behavior we described here, however, is a choice between liked options. We anticipate our findings to be a starting point for delving into neural circuits that govern choosing behavior after prudent comparison rather than intrinsic impulse. Disclosure statement The authors have no conflicts of interest to declare. Acknowledgements The authors thank Drs Zuoren Wang and Sheng Zhang, the Bloomington stock center for fly stocks, Beika Lu, Edward Yao, 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) (2010CB944901, 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).
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