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The accessory gland products (Acps) of male Drosophila cause changes in the behavior and physiology of female flies. Sex peptide (SP) is one of the Acps that initiates many changes including an increase in egg production. The data presented here show that sex peptide overexpression in transgenic female (G-10) flies increases egg production when females are kept on a standard and on high calorie diet, relative to controls not expressing sex peptide. Particularly high increase in egg production observed in G-10 females on high calorie diet suggests that SP overexpression magnifies the female response to caloric uptake. However, on a calorie restriction diet, the fecundity of transgenic females overexpressing sex peptide is lower then control females. On a high caloric diet mating increases early egg production in G-10 and control females, but life-long total egg production is only increased in control females, most likely due to the physiological changes set off by substantial initial egg production in G-10 females.
Mating in Drosophila melanogaster is a complex process, extending beyond a brief physical interaction between males and females to a disparate set of physiological consequences observed in female flies. A negative relationship between reproduction and longevity has been reported for both sexes in a number of experimental animals. Drosophila virgin females and males live longer than mated ones [1-6]. However, the effect of mating on survivorship and fecundity varies with the age of the female fly . Mating early in life causes dramatic increases in egg production and female mortality rate, but if males are removed an initial increase in mortality is partially reversed. During midlife, however, mating does not increase female egg production but does cause an irreversible increase in mortality rate . Although mating decreases male survivorship as well, females experience many other physiological changes that modify their behavior. Post-mating changes in female physiology are caused by the 112 male accessory gland proteins (Acps) that are transferred to females during mating . These proteins and peptides increase the rate of egg production and egg laying, regulate proteolysis, affect immune response, reduce female receptivity to further mating, and decrease survivorship [7-10]. Acps also affect transfer and storage of the sperm and displace sperm from previous matings.
Sex peptide (SP) (Acp70A) was one of the first isolated Acps and its physiological role has been the object of numerous investigations [11, 12]. Injection of isolated SP into virgin females is sufficient to prevent mating and increase egg production . Similarly, overexpression of SP in transgenic female flies decreases their receptivity to matings and causes extrusion of the ovipositor, while absence of SP causes only weak and short response to mating [9, 12, 13]. SP stimulates the corpora allata, endocrine glands, to secrete lipid-like juvenile hormone (JH), which stimulates oogenesis and vitellogenesis . SP also affects the expression of a limited number of genes, increases food intake, and alters immune response and antimicrobial peptide synthesis [15-18].
Similar to reproduction, the level of caloric uptake profoundly affects the physiology and survivorship of a variety of species including flies. Calorie restriction (CR) defined as a decrease in caloric uptake without starvation, decreases reproduction and weight, delays age-related pathophysiology, and increases mobility and survivorship [19-23]. The goal of this investigation was to determine how the overexpression of SP affects egg production when females are kept on different calorie levels. The flies used in experiments were transgenic G-10 flies, in which the SP gene is continuously and ectopically overexpressed in the adult fat body by use of the promoter of a yolk protein enhancer . Thus, the G-10 females continuously synthesize SP in fat body and secrete into the hemolymph. The data presented here show that SP overexpression in females increases egg production when females are kept on standard and high calorie diets but decreases production under CR. Mating increases early egg production in females on a high calorie diet but has no effect on life-long total egg production in SP overexpressing females most likely due to physiological changes that occur when energy resources are shifted from egg production to deal with the effects of mating and Acps.
Standard laboratory corn media as well as food marked as 0.5X, 1.0X and 3.0X were used. The three food levels are standardized as 1.0X being the food that has 100 g/L of sucrose (MP Biomedicals, Inc), 100 g/L of brewer's yeast (MP Biomedicals, Inc) and 20 g/L of agar [19, 20]. 0.5X food contains 50g/L of sucrose and 50 g/L brewer's yeast, 3.0X food contains 300g/L sucrose and 300 g/L brewer's yeast, and both have 20 g/L agar.
The G10 (transformant line G10-3) and cn,ry42 strains were generously provided by Drs. Eric Kubli, Stephen L. Helfand and Claudio Pikienly . The G-10 transgenic flies express sex peptide (SP) constitutively in their fat bodies from where SP is released into the hemolymph. The line was originally generated in Kubli laboratory and described in detail by Aigaki et al. . Briefly, the flies were generated by germline transformation of cn,ry42 strain with construct containing yolk protein (yp1) promoter fused to the SP gene. yp1 drives ectopic expression of SP in the fat body of G-10 flies. Flies were maintained in a humidified temperature-controlled environmental chamber at 25°C (Percival Scientific) on a 12-hour light: dark cycle.
Virgin flies were collected on CO2. Single virgin females, or a single female and a single male of the same age were kept in a plastic vial containing a 0.5X, 1.0X or 3.0X food medium at 25°C in a humidified incubator. Dead males were replaced with the sibling males of the same age. The flies were passed to new vials daily and the number of eggs and age of death for each individual female was recorded.
Generalized estimating equations (GEEs) using STATA 10.0 statistical software were used to test the effects of genotype (G-10, vs. cn, ry), food levels (0.5X, 1.0X and 3.0X) and age [24, 25]. GEEs were also used to determine whether G-10 virgins on 3.0X food produced more or less eggs than cn, ry virgins on 3.0X food over their lifetime. Using a traditional linear model such as the analysis of variance (ANOVA) would have been inappropriate for these data since the distribution of egg production was heavily positively skewed with many instances of zero egg production. The negative binomial link was used as the appropriate link function.
It has been previously reported that injecting or overexpressing sex peptide in female Drosophila increases their egg laying and suppresses their receptivity to further mating . It is known that the caloric content of the food also greatly affects female fecundity. In order to examine the interaction of these two major modifiers on Drosophila fecundity, the daily egg production of female transgenic flies overexpressing sex peptide (G-10) and genetically matched control (cn,ry) flies maintained on food with three different caloric contents were compared. Overexpression of SP ectopically in fat bodies of G-10 flies is driven by yolk protein enhancer (yp1) . The parental flies were grown on standard corn laboratory media and on the day of eclosion a single male and female were placed in a vial with food of one of the three calorie levels: 1.0X, 3.0X and 0.5X, where 1.0X resembles standard laboratory food but does not contain corn [19, 20]. 0.5X food has 50% lower caloric content than 1.0X, and it is considered CR food. Transgenic flies overexpressing sex peptide have a 75% increase in the average egg production on 1.0X food levels compared to the genetic control (G-10=182.2, cn, ry =104.0), Figure 1A, Table 1. The number of eggs laid by the control and experimental flies is lower than the number observed in Canton-S or w1118 wild type stocks previously reported presumably due to their genetic background [20, 23, 26, 27]. In order to maximize egg production we kept G-10 and cn, ry flies on very high calorie food 3.0X. G-10 females on 3.0X food laid 100.1% more eggs compared to 1.0X food levels (G-10: 1.0=182, 3.0X=364.5), Figure 1 D. However, cn, ry flies laid only 15.7% more eggs on 3.0X compared to 1.0X food (1.0X=104, 3.0X=120), Figure 1E. Thus, a total of 302% increase in egg production is observed in G-10 females compared to cn, ry females on 3.0X food, Figure 1 B, Table 1. Generalized Estimating Equations (GEE) analysis reported a statistically significant 3-way age X group X food interaction was found (p<0.001), Supplemental Table 1, that is G-10 flies laid more eggs than controls on 1.0 and 3.0 food. In addition, flies of the same genotype laid more eggs on 3.0X compared to 1.0X food.
We determined the egg laying of G-10 and control flies on a low calorie diet, 0.5X. Control females cn, ry laid 34.0% fewer eggs on 0.5X food compared to 1.0X food (cn, ry, 1.0X= 104, 0.5X=68.6), Figure 1C, D, E, Table 1. G-10 flies on 0.5X food have 89% lower average life-long egg production compared to the G-10 on 1.0X and surprisingly, 70.1% lower compared to control flies on 0.5X food (0.5X: G-10 = 20, cn, ry= 68.6), Figure 1C, D, E, Table 1. It is possible that although these females have increased fecundity compared to the control flies on normal or high calorie diet, additional effects of sex peptide made flies more vulnerable to stressful condition of CR.
Figure 1B indicates that female flies on 3.0X food laid most of their eggs during the first 10 days of life. Females on 1.0X food level produced fewer eggs during their life-time compared to 3.0X, however, they produce eggs longer in life. In order to further examine how egg-laying patterns change with the age of G-10 and cn, ry females we compared the sum of eggs laid in a 10-day period by females on different food levels, Figures 2 A and B, Table 2. The G-10 flies exhibit a much more pronounced difference than control flies in the number of eggs laid on different food in the 10 day period, demonstrating that not only do G-10 flies produce more eggs but they also respond more to different food levels. G-10 flies produce 134.5 % more eggs on 3.0X food compared to 1.0X food during the first period, while cn, ry produce only 42.6 % more on 3.0X compared to 1.0X food (G-10 3.0X 0-10= 233, 1.0X=99.4; cn, ry 3.0X=100.3, 1.0X=70.3), Figure 2 A and B, Table 2. Similarly, control flies exhibit a greater decline in the eggs laid from one time period to another than G-10 flies on the same food levels. For instance, G-10 on 3.0X food levels produce 49 and 89 % fewer eggs in the second and the third periods compared to the first period, while cn, ry have 80% percent fewer eggs produced during the second period and no eggs laid in the third period. Negative binomial regressions found a statistical significant increase in eggs laid by G-10 compared to cn, ry female on 1.0X food during the first three periods and on 3.0X during the first two periods. G-10 females laid significantly fewer eggs than cn, ry on 0.5X food during the first three periods, Supplemental Table 2, 3, 4.
Virgin transgenic G-10 females overexpress SP that increases their egg production. Mated G-10 females receive additional SP and the other Acps provided by males during mating, which affect female fecundity as well. In order to find out how different levels of SP affect egg laying, life-long egg production was determined in females overexpressing SP and control females, both of which were kept as virgins or mated from day 0. Since female fecundity is the highest on a very high calorie diet, cumulative effects would be most easily detected on this type of food thus females were kept on 3.0X food level. Mating changes both the total number and age-specific egg-laying pattern, Figure 3A and B, Table 2. As expected, the number of eggs laid by G-10 virgin females was significantly higher than number of eggs laid by cn, ry females (G-10 = 385.2, cn, ry =80.3; z= 7.47, p<0.001), Table 1, ,2,2, Supplemental Table 5, 6, 7. The total life-long number of eggs in mated control cn, ry females is 33.3% higher than that of virgin females (Mated=120.3, virgins=80.3; z= -5.08, p<0.001). However, the total number of eggs produced in mated G-10 females was 5% lower compared to the virgin G-10 flies (G-10: mated= 364.5, virgins=385.2). During the first 10 days of adult life both G-10 and control females had the greatest increase in egg laying in response to mating. Mated G-10 and cn, ry flies laid 56% and 199% more eggs during the first 10 days after mating compared to the virgin female of the same genotype, respectively, Figure 2B and Table 2. The effect of mating on egg production was less pronounced in G-10 since they already overexpress SP but they still laid more eggs compared to the control cn, ry, flies. However, mated G-10 and cn, ry females produced fewer eggs from 11 to 20 days of age. G-10 produced 31% and cn, ry produce 52% fewer eggs. A similar trend of decreased egg production was observed until the end of life of mated females. Conversely, virgin G-10 and cn, ry females laid more eggs during the second period compared to the first, Figure 3B and Table 2. There are significant differences in age-dependent patterns of egg laying between mated and virgin females in both G-10 and cn, ry, Supplemental Table 6, 7. These data suggests that a combination of sex peptide overexpression and transfer of Apcs during mating increases egg production early in life but has no additional effect on female fecundity. In fact, the mated G-10 females laid fewer eggs during their life-time.
Sex peptide is one of the major components produced by the male accessory glands. SP and other Acps get transmitted during mating into the female reproductive organs . The amino terminal end of SP binds to sperm, which act as a SP carriers during sperm and seminal fluid transfer. After mating, sperm and SP are stored in spermathechae and seminal receptacles for more then a week. SP can then be slowly released from sperm and facilitate prolonged effects by binding with its carboxy terminal end to the sex peptide receptor (SPR) [15, 28]. The expression of SPR is widespread but the number of primary targets of SP is relatively small; only six to eight sensory neurons located in the uterus that co-expressed pickpocket (ppk) and fruitless (fru) [29, 30]. The gene ppk encodes a sodium channel subunit that has a role in mechanosensation; fru is the male- specific transcription factor, which is produced by alternative splicing of the transcript. SP mediates a post-mating response in females by binding to the SPR and by the neurotransmission of that signal to the CNS . Binding of SP to SPR affects the physiology and behavior of the female in several ways: it increases egg production and egg laying, decreases the receptivity of females to further mating, increases food intake and initiates an immune response. It has been shown previously that injection of SP or overexpressing of SP in transgenic female flies increases egg production and decreases the receptivity of females to mating .
Another means to affect female fecundity is by modulating caloric uptake. A number of reports show that although caloric content of the food is important, the ratio between sugar and yeast has a larger effect on survivorship and reproduction . We have previously reported that varying caloric content of the food from 0.5X to 3.0X but keeping the yeast – sugar ratio the same, results in dramatic increase in egg laying . Here three different food levels were used to assess the difference in egg production between females overexpressing sex peptide and controls. Overexpression of SP in the fat body is driven by the yolk protein (yp1) enhancer . SP gets secreted from the fat body into the hemolymph, and can then reach its target SPRs. As expected, transgenic females overexpressing SP (G-10) laid 75% more eggs during their life on 1.0X food compared to the controls. On high caloric 3.0X food G-10 females produced and layed twice as many eggs as they did on 1.0X food. Interestingly, there was only a modest increase of 15% in the egg production observed in control females kept on 1.0X versus 3.0X food. Thus, the total difference in eggs laid between SP overexpressing and control flies on 3.0X food is three fold. Part of the huge increase in egg production observed in G-10 females compared to controls on 3.0X is due to increased food consumption caused by SP . Mated females and females overexpressing SP consume more food than females kept as virgins or females mated with males that do not have SP . Microarray analysis of the abdomens and heads of females overexpressing SP showed increased expression of genes involved in metabolism several hours after mating (17). This metabolic switch is most likely necessary for increased egg production. Consequently, the G-10 female on 3.0X produced and laid the most eggs.
Flies kept on 0.5X food experience many beneficial effects identical to ones described in other species on CR. For instance flies on 0.5X food have decreased weight, increased spontaneous locomotor activity, changes in biochemistry and increased survivorship [20, 23, 29]. The effect of caloric uptake on reproduction has been widely reported. Females on CR suppress their fertility until the conditions improve. Once the food resources become available reproduction is resumed. One unexpected observation is decreased egg production in G-10 females relative to controls on 0.5X food medium. While the level of egg production was decreased in control females 34% on low calorie food levels relative to 1.0X food, G-10 females performed much worse.
Mated control females produced more eggs in comparison to the control females kept as virgin on 3.0X food. The total life-long number of eggs in mated control females is 33.3% higher compared to the virgin controls. Interestingly, mating can still increase early egg production in G-10 females most likely due to the transfer of additional Acps that promote egg production, such as ovulin and AD99. The prohormone ovulin (ASP26Aa), affects egg laying in females during the first day after mating flies by increasing ovulation . DUP99B, made in the ejaculary duct, similarly to SP increases ovulation and oviposition .
In the experiments reported here, mated G-10 females laid significantly more eggs during the first ten days of life compared to the virgin G-10 females. However, after the first ten days, egg production in mated G-10 females diminished relative to virgins to the extent that the total lifelong egg production of the mated females was 5 % less than the virgins. One explanation for the dramatic mating-induced lowering of egg production after the first ten days after mating could be a physiological change to keep the balance between the reproduction, body maintenance and DNA repair. That is, during the first ten day period after eclosion most of the energy is diverted to the extraordinary production of eggs and to combat the toxic effect of the mating itself including the harmful effect of Acps on female physiology. During the second time period there may be less available energy, which has to be shifted from egg production to be used for somatic maintenance and to deal with the effect of mating. We have previously reported that female Drosophila adjust their egg production to cope with age-associated changes and increased frailty . A model of how the calorie content of the food may affect the balance between energy storage, reproduction and somatic maintenance has been described . The data presented here extend those findings by examining the relationships between caloric uptake, mating and SP overexpression on egg production and show a dramatic effect when high caloric intake was combined with SP overexpression. The life-long pattern of egg production suggest a physiological shift to divert the available energy resources from a high-energy expenditure for egg production and egg laying to somatic maintenance and DNA repair in order to deal with the harmful effects of mating and the toxic effect of Acps on female physiology.
I thank Suzanne Kowalski for excellent technical support, Drs. Joseph Jack and Stewart Frankel for critically reading the manuscript, and Drs. Kubli and Aigaki for helpful information and fly stocks. I am grateful to Dr. Daniel J. Denis for expert statistical analysis. This work was supported by grant from the National Institute on Health RO1AG023088.