Abstract
How mating affects male lifespan is poorly understood. Using single worm lifespan assays, we discovered that males live significantly shorter after mating in both androdioecious (male and hermaphroditic) and gonochoristic (male and female) Caenorhabditis. Germline-dependent shrinking, glycogen loss, and ectopic expression of vitellogenins contribute to male post-mating lifespan reduction, which is conserved between the sexes. In addition to mating-induced lifespan decrease, worms are subject to killing by male pheromone-dependent toxicity. C. elegans males are the most sensitive, whereas C. remanei are immune, suggesting that males in androdioecious and gonochoristic species utilize male pheromone differently as a toxin or a chemical messenger. Our study reveals two mechanisms involved in male lifespan regulation: germline-dependent shrinking and death is the result of an unavoidable cost of reproduction and is evolutionarily conserved, whereas male pheromone-mediated killing provides a novel mechanism to cull the male population and ensure a return to the self-reproduction mode in androdioecious species. Our work highlights the importance of understanding the shared vs. sex- and species-specific mechanisms that regulate lifespan.
Introduction
The interplay between the sexes influences an individual’s longevity1-3. Caenorhabditis female lifespan is shortened after mating through receipt of male sperm and seminal fluid4, and separately by exposure to male pheromone5. However, previous studies reported contradictory results on how mating influences male lifespan3,6. Therefore, whether and how male lifespan is affected by prolonged exposure and interactions with females is largely unknown.
The Caenorhabditis genus consists of both androdioecious (male and hermaphroditic) and gonochoristic (male and female) species. In androdioecious species such as C. elegans, the population is dominated by hermaphrodites, which reproduce by self-fertilization. Males are usually very rare (less than 0.2% for the standard lab strain N2) and are produced due to spontaneous X chromosome nondisjunction7,8. Under stressful conditions, more oocytes experience chromosome non-disjunction, thus androdioecious species periodically undergo explosions of male populations. The existence of males in androdioecious species may reduce inbreeding and facilitate adaptation to changing environments9. By contrast, in gonochoristic species such as C. remanei, 50% of the population is male, and females and males must mate to reproduce. The mating efficiency of C. elegans males is very low compared to C. remanei males8. Gonochoristic species females secrete pheromones that attract males10, and have distinct behaviors during mating compared to hermaphrodites11,12. How males in androdioecious and gonochoristic species cope with these different mating situations remains poorly understood. Moreover, the utility of killing females by exposure to male pheromone in gonochoristic populations5 is unclear.
Here we report that after mating, Caenorhabditis males suffer from germline-dependent shrinking and death, just as in the case of mated C. elegans hermaphrodites and C. remanei females4. However, C. elegans males and hermaphrodites have differential sensitivity to male pheromone-dependent toxicity, while C. remanei seem immune to this toxicity, and instead use sex-specific pheromones to identify mates. Thus, androdioecious and gonochoristic species differentially utilize pheromone for mating vs hermaphroditic maintenance, while both species suffer the cost of mating through germline-dependent shrinking and death.
Results
C. elegans males live shorter after mating
C. elegans hermaphrodites shrink up to 30% and live 40% shorter after mating4. We wondered if males also experience such extreme post-mating changes. Traditional lifespan assays are performed using grouped worms; however, grouped males live shorter than solitary males13, which could mask the lifespan shortening effect of mating in males. Therefore, we measured the lifespans of solitary males and single males paired with a single hermaphrodite for 6 days from Day 1 to Day 6 of adulthood. (We used fog-2(q71) worms in our assay; fog-2 males are equivalent to wild-type (N2) males, while fog-2 hermaphrodites are self-spermless14, enabling identification of successful mating.) Mated male lifespan was decreased ~35% compared with the unmated solitary males (Fig. 1A, Table S1), similar to the lifespan decrease of mated hermaphrodites4. Also like females, males shrank after 6 days’ mating; by Day 7, the mated males were 10% smaller than the unmated solitary males control (Fig. 1B,C, Table S2).
Males die faster when paired with a hermaphrodite for a longer period: mating with a hermaphrodite for one day did not affect the lifespan of the male, while 2-3 days’ mating shortened male lifespan by 15%, 4-5 days’ mating reduced their lifespan by 25%, and 6 days’ mating increased the reduction to over 35% (Fig. 1D). By contrast, the number of hermaphrodites paired with the single male during mating had much less effect compared to mating duration (Fig. 1E, Fig. S1A,B). The time at which mating occurs within the reproductive period is also not critical for males’ post-mating lifespan decrease; given the same mating duration, males mated with hermaphrodites for the first three days of adulthood had a similar lifespan decrease as those mated with hermaphrodites during Days 6-8 of adulthood (Fig. 1F).
C. elegans males’ post-mating shrinking and death are germline-dependent
We wondered whether pheromone is required for mating-induced death in males. To distinguish pheromone from a direct mating effect, we tested daf-22(m130) mutants, which are deficient in ascaroside pheromone biogenesis15. Wild-type males still died early post-mating when paired with a daf-22 hermaphrodite for 6 days (Fig. 2A). Likewise, daf-22 mutant males lived shorter after 6 days’ mating (Fig. 2B), indicating that the post-mating lifespan decrease in our single-worm pair lifespan assay is due to mating itself rather than pheromone from either sex.
Elevated germline proliferation is one of the major causes of hermaphrodites’ early death after mating4. We wondered whether this killing mechanism is conserved in males. Adult treatment with the DNA replication inhibitor 5-fluorodeoxyruridine (FUdR) has little effect on lifespan and meiosis at low dosage (50 μM)16, but rapidly blocks germline proliferation in mated hermaphrodites4. When treated with 50 μM FUdR during the three-day mating period, male lifespan was unchanged (Fig. 2C). FUdR treatment also eliminated male post-mating lifespan decrease in our 6 days’ mating regime (Fig. S1C,D). Additionally, lacking the germline prevented both shrinking and death: mating caused neither shrinking nor lifespan decrease in germline-less glp-1(e2141) males (Fig. 2D,E, Fig. S1E). These results suggest that germline-mediated post-mating lifespan regulation is conserved between sexes to a large extent.
We have shown previously that osmotic stress resistance correlates well with shrinking in mated hermaphrodites, whereas fat loss does not account for such shrinking4. Changes of glycogen levels in vivo accurately reflect the osmotic perturbation in the environment17; therefore, we measured the glycogen level using iodine staining, and found that mated wild-type worms lost about 30% of the glycogen storage post-mating in a germline-dependent manner (Fig 2F). The mating-induced glycogen storage decrease and subsequent shrinking is conserved between sexes (Fig. S2).
Vitellogenin dysrégulation contributes to male post-mating death
To further characterize male post-mating death, we performed genome-wide transcriptional analysis of mated and unmated males: we paired a single male with a hermaphrodite for 3.5 days of mating, then picked the males individually from the hermaphrodites on Day 4 for microarray analysis (Fig. S3A). As a control, we collected the same number of age-matched solitary males. 14 genes were significantly up-regulated and 41 were significantly down-regulated (FDR=0%; SAM18; Table S3, Fig. 3A). Genes whose expression decreased in mated males include extracellular proteins (scl-11, scl-12, zig-4) and predicted lipase-related hydrolases (lips-11, lips-12, lips-13). The most enriched gene ontology (GO) categories were ribonucleoside monophosphate biosynthetic/metabolic process and extracellular region for the down-regulated genes, and nutrient reservoir activity and lipid transport for the up-regulated genes (Fig. 3B, Fig. S3B).
Surprisingly, vitellogenins (vit-4, vit-3, vit-5, vit-6, vit-2), which encode yolk protein precursors made in the female/hermaphrodite intestine for transport into developing oocytes19, were the top up-regulated genes in mated males. They were expressed on average 19 times higher in mated males than in solitary unmated males (Table S3). Males normally do not express vit genes, as they produce no oocytes. We confirmed our microarray finding using VIT-2::GFP males: mating induced ectopic expression of VIT-2: :GFP, especially in the anterior intestine in males. Such overexpression was germline-dependent (Fig. 3D, S3D). Overproduction of vitellogenins is deleterious for hermaphrodites: vitellogenins accumulate in the head and body of older hermaphrodites20; long-lived insulin signaling mutants repress vit gene expression21; and knockdown of the vit genes in wild-type hermaphrodites extends lifespan21. The DAE (DAF-16 Associated Element) motif is present in most vit genes, which are also Class 2 DAF-16 genes21. Thus, we tested the function of PQM-1, the DAE-dependent transcription factor22, in male post-mating death. Mated pqm-1(ok485) knockout males lived as long as the unmated control (Fig. 3E), suggesting it is important for post-mating death. The binding motif for UNC-62, a master transcription regulator of vit genes in hermaphrodites23, also emerged in unbiased motif analysis (Fig. 3C). Using RNAi, we found that knocking down unc-62 was sufficient to rescue the lifespan decrease in mated males (Fig. 3F). Thus, the mis-expression of vitellogenins upon mating contributes to post-mating death in males.
Mating-induced early death in males is evolutionarily conserved within Caenorhabditis
Previously, we showed that C. remanei females, like C. elegans hermaphrodites, also shrink and die faster after mating4, suggesting that the mechanisms are evolutionarily conserved in females. Likewise, we found that male C. remanei also lived significantly shorter after mating with a female C. remanei for 6 days (Fig. 4A). However, while female death requires successful cross-progeny production, as C. remanei males do not induce post-mating death of C. elegans hermaphrodites4, C. elegans males died early when mated with a C. remanei female for 6 days (Fig. 4B), suggesting that a component of mating specific and autonomous to the male, rather than a transferred substance or pheromone, is responsible for male death in both species.
Grouped males also have reduced lifespans in C. elegans and C. remanei
When male C. elegans are housed together, they live shorter compared with solitary males13, and the death rate increases with the number of males in a dose-dependent manner13 (Fig. 5A). (This might be the reason a previous report failed to report shortened lifespan of males after mating, because grouped males were used as the control3.) C. elegans male lifespan is very sensitive to male density: just two males together significantly reduced each individual’s lifespan. In a group of eight males, the individual lifespan had a more dramatic 36% decrease compared with the solitary control (Fig. 5A). C. remanei male lifespan was also influenced by male density, although to a lesser degree than C. elegans males (Fig. 5C). C. elegans males tend to form clumps and attempt to mate with each other. By contrast, C. remanei males rarely form clumps, having much reduced male-male interaction13 (Fig. 5A,C insets). We thought such male-male mating attempts might also lead to post-mating lifespan decrease in a germline-dependent manner as we observed in males mated with females. To test this hypothesis, we placed the grouped males and solitary controls on FUdR plates to inhibit germline proliferation. In the presence of FUdR, grouped C. remanei males had no lifespan decrease (Fig. 5D). However, grouped C. elegans males still lived significantly shorter (11% decrease compared with solitary control, p=0.0032, Fig. 5B), indicating that a germline-independent factor also contributes to C. elegans male lifespan reduction when other males are present.
Male pheromone-dependent toxicity leads to reduced lifespan in grouped C. elegans
It was shown previously that C. elegans hermaphrodites can be killed by male pheromone secreted by grouped males5. We wondered whether male pheromone also affects male lifespan. We held 8 daf-22(m130) (pheromone-less) males together, and found that they lived as long as the solitary wild-type males, suggesting that male pheromone kills males (Fig. 5E). Grouped daf-22 males lived just slightly shorter than solitary daf-22 males (Fig. S4A). The remaining lifespan difference can be explained by germline up-regulation due to mating attempts, since daf-22 males also formed clumps (Fig. S4A inset), and this lifespan difference was completely eliminated when the experiment was performed in the presence of FUdR (Fig. 5F). Therefore, in grouped C. elegans males, early death is due to a combination of germline up-regulation and male pheromone. In fact, males are the victims of their own pheromone: the lifespan of daf-22 males was significantly reduced when they were maintained on plates conditioned by only one wild-type male (Fig. 5G, S4B), suggesting that C. elegans males are extremely sensitive to male pheromone-dependent toxicity.
C. elegans and C. remanei have different sensitivity to male pheromone’s toxicity
We wondered whether in a true male/female species, male pheromone-mediated death is also present, and if there are cross-species effects. We confirmed that C. elegans hermaphrodites die early when grown on plates conditioned with a large number of C. elegans males, as shown previously5 (Fig. 6A, 30 males per plate for conditioning). C. elegans hermaphrodites also died early when exposed to C. remanei male pheromone (Fig. 6A). By contrast, multiple trials of C. remanei females on male-conditioned plates failed to reveal any sensitivity to either remanei or elegans male pheromone (Fig. 6B). We then tested the sensitivity of both hermaphrodites and males to low levels of pheromone (8 males per plate for conditioning), and found that C. elegans hermaphrodites were not as sensitive to male pheromone as males were (Fig. 6C). By contrast, both C. remanei males and females were insensitive to low or high amounts of pheromone (Fig. 6D). Thus, C. elegans males are most sensitive to male pheromone-dependent toxicity, C. elegans hermaphrodites have intermediate sensitivity, and C. remanei appear to be immune to male pheromone toxicity (Fig. S5B).
Discussion
Germline activation induces deleterious changes that cause males to die
C. elegans males and hermaphrodites share many post-mating changes. As we found previously for mated females and hermaphrodites4, Caenorhabditis males also experience germline-dependent shrinking, glycogen loss, and death after mating. Germline up-regulation also leads to ectopic expression of vitellogenins, which contributes to the post-mating lifespan decrease in males. Previously, these yolk protein precursors were only noted to be expressed in hermaphrodites, since males do not produce oocytes, which normally take up vitellogenins in females. Mating also induces significant overexpression of vit genes in hermaphrodites24, indicating that vitellogenin expression is closely coupled with mating-induced germline up-regulation in both sexes. Such coupling may be strong enough to overcome the repression of male vitellogenin expression. The striking similarity of germline-dependent post-mating changes in Caenorhabditis males and females suggests that this mechanism is largely conserved between sexes, and may represent an unavoidable cost of reproduction as a result of mating.
Germline-dependent lifespan shortening appears to be conserved across species over large evolutionary distances, as it occurs in all Caenorhabditis species we tested. Male post-mating death is also conserved beyond the Caenorhabditis genus, as Drosophila males die earlier after mating, as well (Partridge and Farquhar 1981). To ask whether a similar phenomenon may also present in human males, we examined >2000 years of historical records of ancient imperial China (210 BC-1908 AD), reasoning that emperors should have had the best medical care and highest standard of living available at the time, and extensive notes regarding the emperors’ behavior are available. Although our analysis is limited by the information provided in historical records in ancient China (e.g., other death-contributing factors such as sexually transmitted diseases cannot be ruled out), we censored unnatural deaths (e.g., killed in war) as we would for C. elegans studies, and controlled for other factors (e.g., extreme alcohol use). We found that those emperors notorious for lifelong, extremely promiscuous sexual behavior lived 35% shorter than their counterparts (34 ± 2 yrs compared with 52 ± 1 yrs, Fig. 7A, Table S4). Furthermore, analysis of father-son pairs to better control for genetic background and environmental influences (they lived in the same era, therefore had the same standard of living and medical care), still revealed a significant decrease in the lifespan of promiscuous emperors (Fig. 7B-D). While it may seem that any comparison between worms and humans in a germline effect on longevity is highly speculative, it was previously noted that the lifespan of Korean eunuchs was significantly longer than the lifespan of non-castrated men with similar socio-economic status25. Together, these results suggest that some aspects of germline-dependent male post-mating death may be evolutionarily conserved.
Male pheromone-induced killing as a strategy to selectively reduce the male population
In addition to the mating-induced lifespan decrease, C. elegans are subject to killing by male pheromone-dependent toxicity, while C. remanei are not. Our study shows that androdioecious and gonochoristic species have different sensitivities to male pheromone. The androdioecious species (C. elegans) males do not appear to use pheromones as efficiently as chemical messengers to facilitate mating, since they are less able to distinguish hermaphrodites’ pheromone from other species’ female or male pheromone; in fact, C. elegans males are even slightly attracted to their own male pheromone, in part explaining their clumping10 (Fig. S5A). On the other hand, male pheromone is very toxic to C. elegans males. Thus, to C. elegans males, pheromones serve primarily as toxins to kill males. By contrast, C. remanei (gonochoristic species) males are extremely attracted by pheromone produced by C. remanei females, even at a low concentration, and are slightly repelled by male pheromone10 (Fig. S5A), but C. remanei are immune to both elegans and remanei male pheromone toxicity (Fig. 6B,D). Thus, the gonochoristic species C. remanei uses pheromones primarily as chemical messengers to locate mates. It is also worth noting that such female pheromone-mediated attraction is completely abolished in the presence of male sperm10. In C. elegans, males are attracted to old, self-spermless hermaphrodites26,27, suggesting that pheromone retains the function as a chemical messenger under some circumstances in C. elegans. However, due to the presence of self-sperm in the hermaphrodites, C. elegans males do not use pheromone as a primary tool to seek young and middle-aged hermaphrodites.
Caenorhabditis species might utilize pheromones in such different ways due to their different modes of reproduction. In the androdioecious species C. elegans, males are normally rare (0.2%), so the chance that any worm he encounters will be a hermaphrodite is very high; thus, there may be less selection pressure to evolve pheromones as chemical messengers to seek out mates. However, periodically there are explosions of male populations in androdioecious species (e.g., under stressful conditions) to allow outcrossing and ensure genetic diversity9. After this period, however, males are more costly to maintain, and there is pressure to return to a primarily hermaphroditic population. It is notable that because C. elegans males are XO, rather than XY, males may have no selfish drive to maintain their own chromosomes. From the perspective of species, using male pheromone as a dose-dependent toxin may be an effective way to cull the male population and ensure the species returns to the selfreproduction mode when the stressful condition has passed. Use of the pheromone as a toxin to kill males may have arisen to aid the return to hermaphroditism, which can also be promoted by increased hermaphroditic progeny production and decreased mating rates28; these factors could also act in tandem with the selected pheromone-dependent killing of males. Hermaphrodite death at high male pheromone concentration (which would happen extremely rarely in nature) might simply be a rather infrequent result of collateral damage, as the hermaphrodites are less sensitive than males to the toxin. Male-specific culling also occurs in species such as Drosophila bifasciata, in which Wolbachia infection leads to the killing of male embryos, suggesting that sex ratio can be controlled through male-killing29. Mathematical modeling shows that selection in C. elegans favors low populations of males30, and our model provides a mechanism for how this may be achieved.
By contrast, the preponderance of males in a 50:50 population, as in the case of C. remanei, makes the use of pheromone as a toxin less likely, as it would cause too much off-target death to be useful for sperm competition. Our cross-species results suggest that remanei male pheromone is toxic to C. elegans, but both C. remanei males and females are immune to both elegans and remanei pheromone (Fig. 6A,B). These results also suggest that the toxic effect of pheromone may not be due to the pheromone itself, but rather to a receptor-mediated sensitivity to pheromone that is specific to C. elegans, with a greater effect in males than in hermaphrodites. Instead, C. remanei pheromone is used to distinguish males from females, an important distinction in 50:50 mixed populations. Like C. elegans, the primary mode of sperm competition in C. remanei appears to involve seminal fluid transfer of factors that cause the mother to die after producing the father’s progeny, before she has a chance to re-mate4, rather than through a pheromone-based mechanism (Fig. 8).
In summary, germline-dependent early death after mating is conserved between sexes and perhaps even across great evolutionarily distances, and is likely due to an unavoidable cost of mating, the result of mated animals ramping up germline proliferation and subsequently exhausting using their own resources as fast as possible to produce the next generation of progeny. The differential use of pheromones as toxins or chemical messengers by males in androdioecious and gonochoristic species demonstrates that they adopt different strategies to compete, mate, and maintain optimal population ratios.
Author Contributions
C.S., A.M.R., and C.T.M. designed experiments. C.S. and A.M.R. performed experiments. C.S. and C.T.M. wrote the paper.
Materials and Methods
Strains
CB4108: fog-2(q71) V
CB4037: glp-1(e2141) III
DR476: daf-22(m130) II
RT130: pwIs23 [vit-2::GFP] (translational fusion)
PB4641: Caenorhabditis remanei
Individual male mating lifespan assays
All the lifespan assays were performed at room temperature (about 20-21°C); except for glp-1 males’ lifespan assays (performed at 25-26°C). 35mm NGM plates were used for all the experiments in this study. 20 μl of OP50 was dropped onto each plate to make a bacterial lawn of ~10 mm diameter. The next day, one synchronized late L4 male and one late L4 hermaphrodite/female were transferred onto each 35 mm NGM plate. For experiments in Fig. 1E, 1F, S1A-B,D, 2C, multiple L4 hermaphrodites were transferred together with one male. One late L4 male of the same age and genotype was transferred onto the control plates. Except for Fig. 2A, fog-2(q71) hermaphrodites were used as the hermaphrodites in the mating assay, because fog-2 hermaphrodites do not have self sperm, thus allowing us to easily detect successful mating (i.e. eggs and progeny on the plates). We only included males that were able to produce progeny in our analysis. However, for the experiments regarding glp-1 males, mating on FUdR, and inter-species cross between C. elegans males and C. remanei females, we included all the males in the analysis. Worms were transferred onto new plates every other day. If the hermaphrodites were lost or bagged, new unmated Day 1 fog-2 hermaphrodites were added as replacement. Males and hermaphrodites/females were kept together for 6 days (unless noted otherwise in the text); afterwards only males were transferred on to newly seeded plates every 2-3 days. For RNAi experiments in Fig. 3F, synchronized eggs were transferred onto NGM plates with RNAi bacteria, late L4 males were transferred and paired with fog-2 L4 hermaphrodites onto NGM plates seeded with OP50 (to eliminate the possible effect on mating efficiency for different RNAi treatments). Two days later, males and hermaphrodites were transferred onto fresh plates seeded with corresponding RNAi bacteria and males were maintained on RNAi bacteria thereafter. When lifespan assays were completed, KaplanMeier analysis with log-rank (Mantel-Cox) method was performed to compare the lifespans of different groups.
Grouped males
35mm NGM plates were used for all the experiments in this study. 20 μl of OP50 was dropped onto each plate to make a bacterial lawn of ~10 mm diameter. The next day, eight synchronized late L4 males were transferred onto each plate. (Two or four males per plate for experiment in Fig. 5A.) One late L4 male of the same age and genotype was transferred onto the control plates. Males were transferred onto fresh plates every two days, when the males were lost or dead, males from other plates were transferred together to make the size of the group stable.
Male-conditioned plates (MCP) setup
Male-conditioned plates for lifespan assays were prepared as previously described5. Briefly, 60 μl of OP50 was dropped onto each 35mm NGM plate to make a bacterial lawn of ~25 mm diameter. Young Day 1 wild-type males (fog-2 males) were transferred onto each plate. Two days later, they were removed and worms for lifespan assays were immediately transferred onto these male-conditioned plates. These male-conditioned plates were being prepared throughout the course of the lifespan assays (Fig. S4B). For the experiments in Fig. 6A,B, 30 males were used for each conditioning plate. For experiments in Fig. 6C,D, 8 males were used for conditioning and for the experiment in Fig. 5G, only 1 male was used for conditioning for each plate.
Body size measurement
Images of live males on 35mm plates were taken daily for the first week of adulthood with a Nikon SMZ1500 microscope. Image J was used to analyze the body size of the worms. The middle line of each worm was delineated using the segmented line tool and the total length was documented as the body length of the worm. T-test was performed to compare the body size differences between groups of males in the same day.
FUdR experiment
FUdR was added to the NGM media to the final concentration of 50 μM. Late L4 males and hermaphrodites were transferred onto NGM+FUdR plates seeded with OP50. Worms were transferred every two days, and were kept on FUdR plates for different period of time (3 days, 6 days or lifetime as indicated by text).
Glycogen staining
Glycogen staining was performed according to a well-described protocol17. Mating of males was set up as previously described. Right before staining, live males of the same group were picked into a M9 droplet with 1M sodium azide on a 3% agarose pad. Immediately after the liquid was dry, the pad was inverted over the opening of a 50g bottle of iodine crystal chips (Sigma) for 1 minute. After the color stained by iodine vapor on the pad disappear (non-specific staining), the worms were immediately imaged by a Nikon microscope. Due to uncontrollable differences, it is hard to compare the staining performed at different times. Thus, worms from the groups of comparison were mounted onto the same pad (separate M9 droplet if there is no visible difference). Image J was used to compare the mean intensity of iodine staining after the background was subtracted. T-test was performed to compare the staining between different groups (on the same pad).
GFP intensity quantification
10-20 worms of each group were imaged by Nikon Ti. Image J was used to measure the mean and the maximum GFP intensity of the whole body area. T-test analysis was performed to compare the GFP intensity of different groups of worms.
Mated males microarrays
We paired a single male with a fog-2 hermaphrodite for about 3.5 days of mating, then picked the males individually from the hermaphrodites on Day 4 for microarray analysis. As a control, solitary males were collected at the same time. About 150 males (on 150 individual 35mm plates) were collected for each condition and replicate. Three biological replicates were performed. RNA was extracted by heat-vortexing method. Two-color Agilent microarrays were used. The detailed steps and analysis were performed according to a previous report31.
Pheromone chemotaxis assay
This assay was modified from a previous assay10. 10 Day 1 virgin C. remanei or C. elegans hermaphrodites were put in 100 μl of M9 buffer at room temperature overnight with shaking. 100 males of either C. elegans or C. remanei were put in 100 μl of M9. The supernatant solutions were then taken for pheromone chemotaxis assay. 60 mm NGM plates (no food) were used for the chemotaxis assay. Two destination spots (supernatant and M9 control) were separated by about 45 mm, the distance from the origin spot to either destination spot is 30mm. Two 1 μl drops of 1M sodium azide were first applied to the destination spots. When dry, a drop of 1 μl M9 or supernatant was separately added onto the destination spots. Then, over 10 young adult (Day 2) males were placed at the origin spot (try to transfer as little bacteria as possible). After 60 minutes, the paralyzed male worms were scored based on their location. The chemotaxis index was calculated as: (#worms at supernatant destination - #worms at control destination)/(#total worms - #worms at origin).
Analysis of Lifespans of Emperors in Imperial China
In ancient China, agriculture was the main source of the country’s wealth. The development of agriculture began in the Neolithic Era (10,000 BC), followed by improvements in the Bronze Age (1000 BC). Late in the Warring states eras (771221 BC), new iron tools were widely adopted, which revolutionized agriculture in China. Ancient China’s economy depended heavily if not solely on agriculture.
Qin Shi Huang (#1 on the list below) was the first emperor to unify China. By that time, agriculture had already been well developed and the basic structure and the quality of civilization did not change much until the late 1800s. Emperors had the best standard of living and medical care at the time, and the living conditions of emperors in Imperial China (220 BC -1911) remained relatively similar (i.e., the best of agricultural civilization) over this period of 2000 years.
To perform our lifespan analysis analogously with the approach we use to assess worm lifespan, we only included emperors who were over 18 years old when they died and those who reigned over 1 year, in order to exclude the cases of puppet emperors (Table S4). Those rows marked by grey on the list indicate that the emperor’s death is unnatural (killed in a war, rebellion, etc); we censored these emperor at the time of death, analogously to how we would censor worms who died unnaturally or disappeared during a lifespan assay. Those highlighted in yellow are emperors with extremely promiscuous sexual behaviors, as documented by official historical records. Those labeled by shaded yellow means they were considered promiscuous but died unnaturally.
The average lifespan of promiscuous emperors was 34 years, which is 35% shorter than the normal emperors’ lifespan (52 years) (Fig. 7, Table S4). It should be noted that these promiscuous emperors were also noted to indulge in excessive alcohol consumption; however, other emperors who were well-known for their lifelong alcohol indulgence were not short-lived (Examples are Yuan Tai Zong #216 on the list, died at 56; Yuan Shi Zu #219 died at 79).
Another case worth noting is Song Gao Zong (#178), who was originally fertile but is reported to have become infertile when he fled south after defeat by his enemies. By the time he reestablished his dynasty in southern China, he was only 24, but was reportedly no longer capable of reproduction; he died at the age of 81. His case may suggest the link between germline signal and lifespan, perhaps in the same manner as the suggested lifespan extension of Korean eunuchs documented by Min, et al. (2012).
Analysis of father-son comparisons
To better control for genetic background and environmental influences, the lifespans of father and son emperors was compared. The reasons we chose to compare father and son instead of emperor and his brothers are twofold: 1) historical records about emperors’ brothers are much less extensive as those of the emperors themselves; 2) most brothers were killed by the emperor (or his ally) to ensure his ascendency and to secure his sovereignty.
Main Figure Legends
Abbreviations and nomenclature in the paper
- C. e.
- C. elegans
- C. r.
- C. remanei
- 1f1m_6d
- “f” stands for hermaphrodite/female, “m” stands for male, the number before f/m suggests the amount of worms on the same 35mm plate. “6d” means mating for 6 days.
- AAA x BBB
- hermaphrodites/females of genotype AAA are mated with males of genotype BBB. (male is always listed after the “x”)
- MCP
- male-conditioned plates
Acknowledgements
We thank the Caenorhabditis Genetics Center (CGC) for strains, Z. Gitai and N. Wingreen for valuable discussions, and members of the Murphy laboratory for critically reading the manuscript. CS is supported by March of Dimes, and AMR by NIH 5T32GM007388-39. CTM is the Director of the Glenn Center for Aging Research at Princeton.