Evolution of “Live fast, die late” life history strategy in Drosophila melanogaster males

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Large males with high energy reserves often adopt ‘live fast, die young’ life-history strategy to maximize fitness compared to small individuals. However, in promiscuous, iteroparous systems, males that live fast and die late have evolutionary advantage. Using two types of Drosophila melanogaster populations- one that have evolved small size as a response to long term selection for faster development and extended longevity (FLJs) and the other that are large ancestral control populations (JBs), we revisit the (i) disposable soma theory, and (ii) live fast, die early life history strategy hypothesis. Contrary to the two hypotheses, the small FLJ males with significantly reduced energy reserves invested heavily in pre-reproductive traits as indicated by significantly higher courtship duration, number of mounting attempts, and comparable copulation duration to JB males. FLJ males also have comparable lifetime fecundity and realized fitness without compromising on longevity, suggesting that FLJ males are adopting a novel “live fast, die late” life history strategy. Biological sciences/Evolution/Experimental evolution Biological sciences/Evolution/Evolutionary theory Figures Figure 1 Figure 2 Figure 3 Introduction According to disposable soma theory, organisms have finite energetic resources to allocate in growth, reproduction, and somatic maintenance 1 . As a result, a tradeoff exists between reproduction and somatic maintenance (affecting longevity). For males, mate attraction, courtship, ejaculate production, and copulation are energetically expensive traits 2 – 5 . Males increase their fitness by mating with multiple females and hence typically allocate more resources to competition for mating 6 , 7 . The combined costs of these reproductive traits are expected to trade-off with longevity 8 . Males with high amount of metabolic resources (i.e., high-condition males) are reported to increase investment in secondary sexual traits- such as sexual displays and are reported to suffer a longevity cost 9 , 10 . The other interpretation emerging from these studies is ‘low-condition males invest little in reproduction related traits and are thus living long- that is, they adopt a live slow and die late life-history strategy. For promiscuous species that exhibit last male advantage, the best life-history strategy in order to maximize Darwinian fitness is to adopt a ‘live fast and die late’ life-history strategy. In this study, we evaluated the veracity of (i) disposable soma theory, and (ii) live fast, die young life history hypothesis by assessing survival and reproduction related traits in males from two types of Drosophila melanogaster populations. The first type are three populations that are under selection for faster development and extended longevity (FLJs) and the other three are their ancestral controls (JBs). The flies from FLJ populations have evolved small size, perhaps due to significantly reduced feeding duration after attainment of the critical size 11 and are akin to low-condition, and possess reduced energy levels (this study) not due to poor diet quality but due to reduced time available for feeding. The JB populations are large with high energy reserves (this study) compared to FLJs, and thus were similar to high-condition due to sufficient feeding time and not due to high quality diet. In our earlier studies, we have shown that the reduction in size of the FLJ flies is not due to starvation or stress like conditions 12 . Although the total feeding duration was significantly reduced in the FLJs compared to JBs, the feeding rates were not different 11 . Further, the gut microbiota in FLJ and JB populations were comparable 13 , suggesting similar food quality during development. Despite having significantly reduced energy reserves, compared to JB males, FLJ males showed significantly higher courtship duration and number of mounting attempts and comparable copulation duration, and were able to trigger identical fecundity profile the female partners, ensuring similar lifetime fecundity and realized fitness. Collectively, these results suggest that FLJ males with reduced energy reserves are investing heavily in energetically costly pre-copulatory traits. Therefore, according to disposable soma theory they are expected to have significantly short lifespan. On the contrary, they have non-significantly higher lifespan compared to JB males. Taken together, the results from this study suggest that FLJ males are evolved a “live fast, die late” life history strategy and thus defy the disposable soma theory 1 . Results Adult size and energy reserves The male adult size at emergence had significantly reduced in the FLJs (196.34 µg/fly) compared to JBs (280.92 µg/fly), (F 1,2 = 57.212, p = 0.001, Fig. 1 a). As, size is often considered as an indicator of resources available to the organism 14 , we estimated the energy levels in the FLJ and JB males by quantifying the whole body carbohydrate, protein and lipid levels; multiplied them by their respective calorific values of 4.2 cal/mg, 4.19 cal/mg and 9.5 cal/mg, summed and averaged over replicate samples to obtain mean energy level per fly for the three replicate populations. The FLJ males (0.637 calories/fly) had significantly reduced energy levels (F 1,2 = 7.946, p = 0.047, Fig. 1 b) compared to JB males (1.054 calories/fly). Pre-copulatory traits: Since, selected males have reduced energy reserves at emergence and are under selection for extended adult longevity, they should have reduced their investment in early life pre-copulatory traits. To test this hypothesis, we assessed three pre-copulatory traits. All the data failed the normality test, therefore Mann-Whitney Test was performed to compare the traits. Courtship duration of FLJ males (546.233 ± 145.535 sec) was significantly higher (Mann- Whitney U = 306, p = 0.016, Fig. 2 a) than JB males (453.433 ± 159.851 sec). Mounting attempts of FLJ males (19.6 ± 5.075) were also significantly higher (Mann- Whitney U = 225.5, Fig. 2 b) than JB males (6.266 ± 1.717). However, copulation duration was comparable (Mann- Whitney U = 375.5, p = 0.316, Fig. 2 .c) between FLJ (1065 ± 65.003 sec) and JB males (1046.464 ± 44.846 sec). These data suggest that the selected males are investing heavily in pre-copulatory traits despite having reduced energy. Male lifetime fecundity, fertility and longevity: In order assess, the investment of energy in pre-copulatory traits, translates to ultimate fitness parameters-fecundity and fertility, we established 30 male:female pairs for each population type and counted the number of eggs laid by the pair over their life time and ascertained the viability of eggs at weekly intervals. A common female type was used as a breeding partner so as to minimize the confounding effect of female partner. Life-time fecundity data was subjected to one way ANOVA with selection as fixed factor and replicate blocks as random factor 15 . Life-time fecundity elicited by FLJ males was comparable to that by JB males (F 1,2 = 5.344, p = 0.081, Fig. 3 a). Two-way ANOVA was performed for male fertility with selection and days as a fixed factors, and replicate blocks as a random factor. FLJ male fertility (percent eclosion) was comparable with JB male fertility (F 1,2 = 1.360, p = 0.363, Fig. 3 c). Census records were maintained till the death of all assay flies to assess the longevity of males. We have compared the survival probabilities of males by using Kaplan-Meier Analysis followed by log-rank (Mantel-Cox) test. The selected males had non-significantly higher survival probability than control males (c 2 = 2.564, p = 0.109, Fig.3d). Discussion Adult body size is shown to reduce as a response to the selection for faster development 11 , 15 – 17 . Similarly, FLJ males have evolved small size as a correlated response to selection for faster pre-adult development as indicated by significantly lower dry weight (Fig. 1 a). Size is often considered as an indicator of resources available to the organism 14 that are utilized for adult life history traits throughout the life 8 . Hence, we estimated the total energy reserves (calories) in FLJ and JB males and found them to be significantly reduced in FLJ males (Fig. 1 b). Since, selected males have reduced energy and are under selection for extended reproductive lifespan as adults, they should have reduced their investment in pre-copulatory traits during their early life. On the contrary, the FLJ males are investing heavily in pre-copulatory traits despite having reduced energy levels (Fig. 2 a-c), and were able to achieve comparable realized fitness (Fig. 3 a-c). According to disposable soma theory, the FLJ males should have compromised on their longevity as they have drastically reduced energy levels and are investing heavily in pre-copulatory behaviours in order to achieve comparable realized fitness. However, FLJ males had non-significantly higher survival probability compared to JB males (Fig. 3 d). This non-significantly higher survival was not due to early-death of the female partner (data not shown). Energy reserves available to individuals at the time of emergence as imago determine their life-history strategies 8 , as their energy levels do not change post emergence due to their post-mitotic nature 18 . Besides, most insects are promiscuous and iteroparous. Hence, males are reported to invest heavily in pre- and post-copulatory traits to access females and maximize their fitness. However, those that invest heavily in reproduction related traits are reported to pay a cost in terms of reduced longevity and lost future reproduction opportunity 7 , 8 , 19 . This is popularly referred to as ‘live fast, die young’ life-history strategy. A number of studies have shown that the ‘live fast, die young’ strategy is adopted by individuals with abundant metabolic reserves, called the high-condition (a.k.a. large) individuals. The alternative life-history strategy is the ‘live slow, die late’ strategy adopted by the low-condition (a.k.a. small) individuals. The two strategies might co-exist in populations due to the promiscuous and iteroparous nature of reproduction. However, the best evolutionary strategy would be ‘live fast and die late’ due to the last male advantage 20 in systems with promiscuity and iteroparity. The results from the present study show that populations of Drosophila melanogaster selected for fast development and extended longevity (FLJ) were significantly small at emergence (Fig. 1 a), had significantly low energy levels (Fig. 1 b), invested significantly more in pre-copulatory behaviours (Fig. 2 a, 2 b) and had comparable copulation duration (Fig. 2 c), elicited comparable life-time fecundity (Fig. 3 a), fecundity profile (Fig. 3 b) and realized Darwinian fitness (Fig. 3 c) and longevity (Fig. 3 d). These results clearly show that the energy depleted FLJ flies follow a ‘live fast, die late’ life history strategy and lay to rest the universality of disposable soma hypothesis. Materials And Methods Stock Populations: A total of six Drosophila melanogaster populations were used in this study. Three of them were control populations (JBs). The other three populations were derived from the control populations by subjecting them to simultaneous selection for faster pre-adult development and extended longevity (FLJs - Faster developing, late reproducing, derived from JBs). These populations were maintained at standard laboratory conditions (SLC) of 25 ± 1º C, 70 ± 5% relative humidity, and 24:0::L:D in Powers Scientific Inc. USA, environmental chambers and reared on a standard Banana–Jaggery media (SM) 21 . JBs were cultured in 40 glass vials (9.5 × 2.3 cm) per replicate population with density of ~60 eggs per vial containing 6 ml SM. These 40 vials were incubated at SLC for 12 days from the day of egg collection (ECD). On the 12 th day, these flies were transferred to the pre-labeled plexiglass cages (25 cm × 20 cm × 15 cm) containing ad libitum SM. Every alternative day, the food plates were replaced with fresh SM plates. On the 18 th day from the ECD, a fresh SM plate added with live yeast-acetic acid paste in cages. The yeast supplemented plate was replaced with fresh uncontaminated SM media plate cut into two halves (in order to increase vertical surface area) on 20 th day at 18:00 hours. The cut plates were removed from the population cages on 21 st day at 9:00 hours, and eggs were counted under Zeiss Stemi DV4 stereo zoom microscope and dispensed into 40 fresh media vials at a density of ~60/ 6 mL media vial for starting the next generation cycle. The maintenance regime of FLJs was similar to JBs except that for culturing FLJs, 160 vials were maintained per replicate population with a moderate density of ~70 eggs per vial and only the early emerging 15-20 flies from each vial were transferred to the plexiglass cages for 130 generations. In order to avoid adult overcrowding, each of the FLJs population was maintained in sister cages. The cages were monitored for mortality while providing fresh SM plates every alternate day. On noticing 50% mortality (visual assessment) in any cage, all cages were provided with fresh SM plate added with live yeast-acetic acid paste for ~2 days. The yeast supplemented plates were replaced with fresh uncontaminated SM plate cut into two halves on the 3 rd from the day of 50% mortality assessment. The cut plates were removed from cages after 1 hour and eggs collected and dispensed into 160 vials at a density of ~70 eggs/ 6 mL media vial. To avoid independent evolution in sister cages, eggs from them were mixed and redistributed in vials. Beyond 130 generations, only 80 vials per replicate populations were maintained and early emerging 25-30 flies from each vial were transferred to the plexiglass cages. The pre-adult duration of FLJs was 7½ days as against 9½ days in their ancestral control JB populations 11,22,23 . The FLJ adults are significantly smaller compared to the JB adults 11,16,22 Generation of assay flies: At the time of starting this study, JB and FLJ had been through 433 and 219 generations, respectively. Before starting each experiment, the JB and FLJ populations were passed through common rearing conditions for one generation to eliminate non–genetic parental effects. Eggs were collected in 40 vials per population at a density of ~50 eggs in 6mL SM in each vial and incubated at SLC. All the flies that eclosed from these vials at the end of the 10 th day for FLJ and on the 12 th day for JB from the ECD were transferred to plexiglass cages with sterile SM food plates. These flies were referred to as standardized flies. The assay flies were generated from the standardized flies and egg collection was staggered by the developmental time difference to obtain assay flies of similar-age for experiments 22,24 . After red eyes became visible in pupae, vigil checks were carried-out at every 4 hour interval. Emerged flies were sorted according to their sex and maintained in unisex vials with 6 mL SM till used in other experiments. Flies were sorted until 80% of the flies had eclosed. The virgin flies of a given gender were pooled before being used in various assays. The purpose of this study was to assess the ‘live fast, die young’ hypothesis in males. Hence, in order to minimize the influence of the females we have used the females from the respective JB population in all the assays. Two types of mating pairs were set up: JB female × FLJ male and JB female × JB male. Quantification of Energy reserves at emergence: Five samples of 10 virgin male flies per replicate population of each selection type were prepared for estimation of carbohydrates, proteins and lipids. 1) Carbohydrates estimation: Total carbohydrates was assayed using Anthrone method 25 with slight modifications. Virgin flies were homogenized in 200μl of 0.1M PBS. Samples were centrifuged at 12,000 rpm for 10 minutes at 4 °C. 5x dilution was prepared by adding Milli-Q water to samples. 800μl of anthrone reagent (Merck- CAS No. 90-44-8) was added, and the reaction mixture was boiled at 90 °C in a water bath for 18-20 minutes. Samples were cooled to room temperature and were loaded into the 96-well plate. Each biological sample was plated in triplicate. Absorbance was measured at 625nm using ELISA plate reader. The standard glucose curve was used to estimate total carbohydrates. 2) Protein estimation: Protein estimation was performed using Pierce tm BCA Protein Assay Kit (Thermo Scientific, Catalog no.-23227) following manufacturer protocol. Virgin flies were homogenized in 300μl 0.1M PBS. To make the working reagent, 50 parts of BCA reagent A with 1 Part of BCA reagent B were mixed (50 BCA reagent A: 1 BCA reagent B). Standard and samples were plated on 96-well plates and 200μl of working reagent was added to both. Each biological sample was plated in triplicate. The plate was agitated on a plate shaker for 30 seconds and incubated for 30 minutes at 30 °C. Absorbance was taken at 562nm by using ELISA plate reader. Bovine serum albumin was used to make a standard curve. 3) Lipid estimation: Virgin flies were used to obtain the fresh weight of the flies using microbalance (Model No. CM11, Citizen). Samples were stored in glass vials and dried in hot air oven at 70 °C for 36 hours and weighed again to obtain the dry weight. Flies were transferred to pre-labelled 1.5 ml micro centrifuge tubes. Diethyl ether was used to extract ether-soluble lipids by following the protocol of Handa et. al., 2014 with few modifications 16 . Lipids were extracted for over a duration for 48 hours with three ether changes with 12 hours interval. After the last ether change, flies oven dried at 47 °C for ~2 hours and weight was again reassed to obtain lipid-free weight of the flies. The difference between lipid-free weight and dry weight of the flies was used to calculate lipid content. Energy estimation: The carbohydrate, protein and lipid data were multiplied by their calorific values 4.2 cal/mg, 4.19 cal/mg and 9.5 cal/mg respectively 26 . The resulting data was summed and averaged per replicate population in order to obtain the total energy levels of flies. Reproduction related traits assay: 1) Pre-copulatory traits: Virgin male and female flies were aged for 3 days before pairing them for assessment of pre-copulatory behavioural traits- total courtship duration (sec) and number of mounting attempts, and copulation duration (sec). Each male-female pair was introduced into mating chamber of 1.7 cm height × 2 cm diameter dimension. All activities performed by the pair was video recorded till the disengagement post copulating pair or for a maximum duration of 1 hr in case of pairs that did not copulate 27 . Pre-copulatory behaviour data were extracted by viewing video recordings. Pre-copulatory traits and copulation duration were assessed on only one replicate population each of FLJ and JB. 2) Life-time fecundity, fecundity profile and realized fitness traits: Individual male-and female pair were transferred to sterile 3mL SM vials. Thirty such pairs per treatment per replicate population were set-up and held in Powers Scientific Incubators USA, environmental chambers. The pairs were flipped into fresh SM vials every 24 hours. Eggs laid in the preceding 24 hour period were counted under Zeiss Stemi DV4 stereo zoom microscope. This process was carried out till the death of all flies. The total life-time fecundity and fecundity profile were obtained from this data. In order to assess the realized fitness, we incubated egg vials at pre-determined adult ages and counted the number of emerging adults to estimate the % viability. We incubated 15 vials containing maximum number of eggs during the first half of the experimental duration and nearly all vials during the latter half the experimental. Longevity: Using the mortality data from the reproduction related traits assay set-up, we constructed Kaplan-Meir survival probability curves. Statistical analyses: All the traits data was subjected to Shapiro-Wilk normality test. All data that passed the normality test, were subjected to Bartlett’s test of homogeneity 13 . The energy equivalents and life-time fecundity passed the normality test and hence we adopted one-way ANOVA model with selection as a fixed factor and replicate block as a random factor. The pre-copulatory traits data failed the normality test, hence were subjected to non-parametric, Mann Whitney U test. Male fertility data passed the normality test, hence was subjected to two-way ANOVA with selection and days as fixed factors, and replicate blocks as random factor. The survival probabilities of males were assessed using non-parametric Kaplan Meier analysis followed by log-rank (Mantel-Cox) test using Graphpad version 8. Declarations Funding: This research was supported by Institute of Eminence (IOE), University of Delhi grant (Ref. No./IOE/2023-24/12/FRP) to MS. We thank Council for Scientific & Industrial Research (CSIR) for research fellowship to AKF (File No. 09/0045(12766)/2021-EMR-1). We also thank University Grants Commission (UGC) for research fellowship to AY (Ref. No. 211610167457) and to NKS (Ref. No. 19/06/2016(i)EU-V-346377). Author contributions: Conceptualization: AKF, MS Methodology, investigation, validation: AKF, NKS, NR, MK, AY Funding acquisition and supervision: MS Writing – original draft: AKF Writing – review & editing: AKF, NKS, MS Competing interests: The authors declare no competing interest. Data and materials availability: Data is available as supplementary sheets. References Kirkwood, T. B. L. Evolution of ageing. Nature 270, 301–304 (1977). Chung, M.-H. J., Jennions, M. D. & Fox, R. J. Quantifying the costs of pre- and postcopulatory traits for males: Evidence that costs of ejaculation are minor relative to mating effort. Evolution Letters 5, 315–327 (2021). Dewsbury, D. A. Ejaculate Cost and Male Choice. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4516780","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":311379963,"identity":"dcb73af4-c003-4360-a9e1-b89b3e727c64","order_by":0,"name":"Mallikarjun Shakarad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACAwYGxgOMDUAWO4gwsCBKCwNEC88BEFeCFC0SCSA+EVrM2ZsPHPi5gyFafubzqxt+FEgw8Ld3J+DVYtlzLOFg7xmG3A23c8pu9gAdJnHm7Ab8DruRY3CAtw2oRTon7QYPUIuBRC4BLffffzj4F6hl/swzaTf/EKXlBg/DYZAtDTfYj90myhbLnjSDw7JngMrO5LDdljGQ4CHoF3P2ww8fvt1hkzu//fizm2/+2Mjxt/fi1wIFoOjgAcURAw8xymGA/QEpqkfBKBgFo2AEAQCA304z8Mp35wAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Zoology, University of Delhi","correspondingAuthor":true,"prefix":"","firstName":"Mallikarjun","middleName":"","lastName":"Shakarad","suffix":""},{"id":311379964,"identity":"c1e1062b-fa29-4290-9297-8e3189f27c36","order_by":1,"name":"Abhishek Farand","email":"","orcid":"","institution":"Department of Zoology, University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Farand","suffix":""},{"id":311379965,"identity":"35d79e77-0bb6-419b-85bd-cdece184d559","order_by":2,"name":"Nidhi Shrivastava","email":"","orcid":"","institution":"Molecular Mechanisms of Symbiosis Laboratory, Institute of Environmental Sciences, Jagiellonian University, Krakow 30-387, Poland","correspondingAuthor":false,"prefix":"","firstName":"Nidhi","middleName":"","lastName":"Shrivastava","suffix":""},{"id":311379966,"identity":"80bab5e1-eaaa-4ed2-ba3f-189abeb8dbf3","order_by":3,"name":"Neha Rauhila","email":"","orcid":"","institution":"Department of Zoology, University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Neha","middleName":"","lastName":"Rauhila","suffix":""},{"id":311379967,"identity":"cfaee4ae-fae2-496d-b9fa-fbc03c6dbf58","order_by":4,"name":"Meenakshi Khati","email":"","orcid":"https://orcid.org/0009-0005-1758-4289","institution":"Department of Zoology, University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Meenakshi","middleName":"","lastName":"Khati","suffix":""},{"id":311379968,"identity":"e9bf36ce-84f7-4acc-b774-b4a387c83432","order_by":5,"name":"Abhishek Yadav","email":"","orcid":"","institution":"Department of Zoology, University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Yadav","suffix":""}],"badges":[],"createdAt":"2024-06-02 11:15:15","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4516780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4516780/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58748307,"identity":"f4c3087f-38d3-4883-a1b3-60ba54187c8d","added_by":"auto","created_at":"2024-06-20 15:22:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45444,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Average dry weights (mg/fly) of JB and FLJ males. (b) Average energy (calories/fly) in JB and FLJ males at emergence. Data presented as mean ± sem. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4516780/v1/176923e01c6f7716299e18e2.png"},{"id":58748309,"identity":"602e2f04-a762-426d-b103-a41dc370a1c2","added_by":"auto","created_at":"2024-06-20 15:22:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66895,"visible":true,"origin":"","legend":"\u003cp\u003eFLJ males show significantly higher pre-copulatory behavioual traits. (a) Courtship duration is significantly higher in FLJ males (\u003cem\u003ep\u003c/em\u003e= 0.016). (b) Mounting attempts are significantly higher in FLJ males (\u003cem\u003ep\u003c/em\u003e = 0.0004). (c) Copulation duration is comparable between JB and FLJ males (\u003cem\u003ep\u003c/em\u003e= 0.316). Data is presented as a box-and-whisker plot to show variance. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4516780/v1/a3e90fb4174b5943aecde4e8.png"},{"id":58747407,"identity":"d99bc858-bd62-45a4-896a-31637ae802be","added_by":"auto","created_at":"2024-06-20 15:14:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90939,"visible":true,"origin":"","legend":"\u003cp\u003eThe life-time fecundity and effective realized fitness were comparable between FLJ and JB males. (a) Life-time fecundity of JB females when paired either with JB or FLJ male (\u003cem\u003ep \u003c/em\u003e= 0.081). (b) Similar and over-lapping 3-day running averages of number of eggs laid by JB female when paired either with JB and FLJ male. (c) Percent eclosion (egg viability) of JB female when paired either with JB and FLJ male (\u003cem\u003ep \u003c/em\u003e= 0.363). (d) Percent survival of FLJ and JB males when paired with JB females. FLJ males have non-significantly higher percent survival (\u003cem\u003ep \u003c/em\u003e= 0.109). Color code, Black- JB*JB and purple-FLJ*JB.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4516780/v1/0fcb8691341177ffa03ab3ea.png"},{"id":58749644,"identity":"52c7fda0-0ca5-48f1-be9a-aae9b7552a4b","added_by":"auto","created_at":"2024-06-20 15:38:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":607333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4516780/v1/a31e969b-c5c7-4cc3-acca-394b78a21baf.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Evolution of “Live fast, die late” life history strategy in Drosophila melanogaster males","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to disposable soma theory, organisms have finite energetic resources to allocate in growth, reproduction, and somatic maintenance\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As a result, a tradeoff exists between reproduction and somatic maintenance (affecting longevity). For males, mate attraction, courtship, ejaculate production, and copulation are energetically expensive traits\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Males increase their fitness by mating with multiple females and hence typically allocate more resources to competition for mating\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The combined costs of these reproductive traits are expected to trade-off with longevity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Males with high amount of metabolic resources (i.e., high-condition males) are reported to increase investment in secondary sexual traits- such as sexual displays and are reported to suffer a longevity cost\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The other interpretation emerging from these studies is \u0026lsquo;low-condition males invest little in reproduction related traits and are thus living long- that is, they adopt a live slow and die late life-history strategy. For promiscuous species that exhibit last male advantage, the best life-history strategy in order to maximize Darwinian fitness is to adopt a \u0026lsquo;live fast and die late\u0026rsquo; life-history strategy.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the veracity of (i) disposable soma theory, and (ii) live fast, die young life history hypothesis by assessing survival and reproduction related traits in males from two types of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e populations. The first type are three populations that are under selection for faster development and extended longevity (FLJs) and the other three are their ancestral controls (JBs). The flies from FLJ populations have evolved small size, perhaps due to significantly reduced feeding duration after attainment of the critical size\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and are akin to low-condition, and possess reduced energy levels (this study) not due to poor diet quality but due to reduced time available for feeding. The JB populations are large with high energy reserves (this study) compared to FLJs, and thus were similar to high-condition due to sufficient feeding time and not due to high quality diet. In our earlier studies, we have shown that the reduction in size of the FLJ flies is not due to starvation or stress like conditions\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although the total feeding duration was significantly reduced in the FLJs compared to JBs, the feeding rates were not different\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Further, the gut microbiota in FLJ and JB populations were comparable\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, suggesting similar food quality during development. Despite having significantly reduced energy reserves, compared to JB males, FLJ males showed significantly higher courtship duration and number of mounting attempts and comparable copulation duration, and were able to trigger identical fecundity profile the female partners, ensuring similar lifetime fecundity and realized fitness. Collectively, these results suggest that FLJ males with reduced energy reserves are investing heavily in energetically costly pre-copulatory traits. Therefore, according to disposable soma theory they are expected to have significantly short lifespan. On the contrary, they have non-significantly higher lifespan compared to JB males. Taken together, the results from this study suggest that FLJ males are evolved a \u0026ldquo;live fast, die late\u0026rdquo; life history strategy and thus defy the disposable soma theory\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAdult size and energy reserves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe male adult size at emergence had significantly reduced in the FLJs (196.34 \u0026micro;g/fly) compared to JBs (280.92 \u0026micro;g/fly), (F\u003csub\u003e1,2\u003c/sub\u003e = 57.212, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). As, size is often considered as an indicator of resources available to the organism\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we estimated the energy levels in the FLJ and JB males by quantifying the whole body carbohydrate, protein and lipid levels; multiplied them by their respective calorific values of 4.2 cal/mg, 4.19 cal/mg and 9.5 cal/mg, summed and averaged over replicate samples to obtain mean energy level per fly for the three replicate populations. The FLJ males (0.637 calories/fly) had significantly reduced energy levels (F\u003csub\u003e1,2\u003c/sub\u003e = 7.946, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) compared to JB males (1.054 calories/fly).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePre-copulatory traits:\u003c/h2\u003e\n \u003cp\u003eSince, selected males have reduced energy reserves at emergence and are under selection for extended adult longevity, they should have reduced their investment in early life pre-copulatory traits. To test this hypothesis, we assessed three pre-copulatory traits. All the data failed the normality test, therefore Mann-Whitney Test was performed to compare the traits. Courtship duration of FLJ males (546.233 \u0026plusmn; 145.535 sec) was significantly higher (Mann- Whitney U\u0026thinsp;=\u0026thinsp;306, p\u0026thinsp;=\u0026thinsp;0.016, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) than JB males (453.433 \u0026plusmn; 159.851 sec). Mounting attempts of FLJ males (19.6 \u0026plusmn; 5.075) were also significantly higher (Mann- Whitney U\u0026thinsp;=\u0026thinsp;225.5, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) than JB males (6.266 \u0026plusmn; 1.717). However, copulation duration was comparable (Mann- Whitney U\u0026thinsp;=\u0026thinsp;375.5, p\u0026thinsp;=\u0026thinsp;0.316, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.c) between FLJ (1065 \u0026plusmn; 65.003 sec) and JB males (1046.464 \u0026plusmn; 44.846 sec). These data suggest that the selected males are investing heavily in pre-copulatory traits despite having reduced energy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eMale lifetime fecundity, fertility and longevity:\u003c/h2\u003e\n \u003cp\u003eIn order assess, the investment of energy in pre-copulatory traits, translates to ultimate fitness parameters-fecundity and fertility, we established 30 male:female pairs for each population type and counted the number of eggs laid by the pair over their life time and ascertained the viability of eggs at weekly intervals. A common female type was used as a breeding partner so as to minimize the confounding effect of female partner. Life-time fecundity data was subjected to one way ANOVA with selection as fixed factor and replicate blocks as random factor\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Life-time fecundity elicited by FLJ males was comparable to that by JB males (F\u003csub\u003e1,2\u003c/sub\u003e = 5.344, p\u0026thinsp;=\u0026thinsp;0.081, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Two-way ANOVA was performed for male fertility with selection and days as a fixed factors, and replicate blocks as a random factor. FLJ male fertility (percent eclosion) was comparable with JB male fertility (F\u003csub\u003e1,2\u003c/sub\u003e = 1.360, p\u0026thinsp;=\u0026thinsp;0.363, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eCensus records were maintained till the death of all assay flies to assess the longevity of males. We have compared the survival probabilities of males by using Kaplan-Meier Analysis followed by log-rank (Mantel-Cox) test. The selected males had non-significantly higher survival probability than control males (c\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 2.564, p = 0.109, Fig.3d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdult body size is shown to reduce as a response to the selection for faster development\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Similarly, FLJ males have evolved small size as a correlated response to selection for faster pre-adult development as indicated by significantly lower dry weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Size is often considered as an indicator of resources available to the organism\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e that are utilized for adult life history traits throughout the life\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Hence, we estimated the total energy reserves (calories) in FLJ and JB males and found them to be significantly reduced in FLJ males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eSince, selected males have reduced energy and are under selection for extended reproductive lifespan as adults, they should have reduced their investment in pre-copulatory traits during their early life. On the contrary, the FLJ males are investing heavily in pre-copulatory traits despite having reduced energy levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c), and were able to achieve comparable realized fitness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). According to disposable soma theory, the FLJ males should have compromised on their longevity as they have drastically reduced energy levels and are investing heavily in pre-copulatory behaviours in order to achieve comparable realized fitness. However, FLJ males had non-significantly higher survival probability compared to JB males (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This non-significantly higher survival was not due to early-death of the female partner (data not shown).\u003c/p\u003e \u003cp\u003eEnergy reserves available to individuals at the time of emergence as imago determine their life-history strategies\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, as their energy levels do not change post emergence due to their post-mitotic nature\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Besides, most insects are promiscuous and iteroparous. Hence, males are reported to invest heavily in pre- and post-copulatory traits to access females and maximize their fitness. However, those that invest heavily in reproduction related traits are reported to pay a cost in terms of reduced longevity and lost future reproduction opportunity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This is popularly referred to as \u0026lsquo;live fast, die young\u0026rsquo; life-history strategy. A number of studies have shown that the \u0026lsquo;live fast, die young\u0026rsquo; strategy is adopted by individuals with abundant metabolic reserves, called the high-condition (a.k.a. large) individuals. The alternative life-history strategy is the \u0026lsquo;live slow, die late\u0026rsquo; strategy adopted by the low-condition (a.k.a. small) individuals. The two strategies might co-exist in populations due to the promiscuous and iteroparous nature of reproduction. However, the best evolutionary strategy would be \u0026lsquo;live fast and die late\u0026rsquo; due to the last male advantage\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e in systems with promiscuity and iteroparity. The results from the present study show that populations of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e selected for fast development and extended longevity (FLJ) were significantly small at emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), had significantly low energy levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), invested significantly more in pre-copulatory behaviours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and had comparable copulation duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), elicited comparable life-time fecundity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), fecundity profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and realized Darwinian fitness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and longevity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These results clearly show that the energy depleted FLJ flies follow a \u0026lsquo;live fast, die late\u0026rsquo; life history strategy and lay to rest the universality of disposable soma hypothesis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStock Populations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of six \u003cem\u003eDrosophila melanogaster\u0026nbsp;\u003c/em\u003epopulations were used in this study. Three of them were control populations (JBs). The other three populations were derived from the control populations by subjecting them to simultaneous selection for faster pre-adult development and extended longevity (FLJs - Faster developing, late reproducing, derived from JBs). These populations were maintained at standard laboratory conditions (SLC) \u0026nbsp;of 25 \u0026plusmn; 1\u0026ordm; C, 70 \u0026plusmn; 5% relative humidity, and 24:0::L:D in Powers Scientific Inc. USA, environmental chambers and reared on a standard Banana\u0026ndash;Jaggery media (SM)\u003csup\u003e21\u003c/sup\u003e. JBs were cultured in 40 glass vials (9.5 \u0026times; 2.3 cm) per replicate population with density of ~60 eggs per vial containing 6 ml SM. These 40 vials were incubated at SLC for 12 days from the day of egg collection (ECD). On the 12\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday, these flies were transferred to the pre-labeled plexiglass cages (25 cm \u0026times; 20 cm \u0026times; 15 cm) containing ad libitum SM. Every alternative day, the food plates were replaced with fresh SM plates. On the 18\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday from the ECD, a fresh SM plate added with live yeast-acetic acid paste in cages. The yeast supplemented plate was replaced with fresh uncontaminated SM media plate cut into two halves (in order to increase vertical surface area) on 20\u003csup\u003eth\u003c/sup\u003e day at 18:00 hours. The cut plates were removed from the population cages on 21\u003csup\u003est\u003c/sup\u003e day at 9:00 hours, and eggs were counted under Zeiss Stemi DV4 stereo zoom microscope and dispensed into 40 fresh media vials at a density of ~60/ 6 mL media vial for starting the next generation cycle.\u003c/p\u003e\n\u003cp\u003eThe maintenance regime of FLJs was similar to JBs except that for culturing FLJs, 160 vials were maintained per replicate population with a moderate density of\u0026nbsp;~70 eggs per vial and only the early emerging 15-20 flies from each vial were transferred to the plexiglass cages for 130 generations. In order to avoid adult overcrowding, each of the FLJs population was maintained in sister cages. The cages were monitored for mortality while providing fresh SM plates every alternate day. On noticing 50% mortality (visual assessment) in any cage, all cages were provided with fresh SM plate added with live yeast-acetic acid paste for ~2 days. The yeast supplemented plates were replaced with fresh uncontaminated SM plate cut into two halves on the 3\u003csup\u003erd\u003c/sup\u003e from the day of 50% mortality assessment. The cut plates were removed from cages after 1 hour and eggs collected and dispensed into 160 vials at a density of ~70 eggs/ 6 mL media vial. To avoid independent evolution in sister cages, eggs from them were mixed and redistributed in vials. Beyond 130 generations, only 80 vials per replicate populations were maintained and early emerging 25-30 flies from each vial were transferred to the plexiglass cages. The pre-adult duration of FLJs was 7\u0026frac12; days as against 9\u0026frac12; days in their ancestral control JB populations\u003csup\u003e11,22,23\u003c/sup\u003e . The FLJ adults are significantly smaller compared \u0026nbsp;to the JB adults\u003csup\u003e11,16,22\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of assay flies:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of starting this study, JB and FLJ had been through 433 and 219 generations, respectively. Before starting each experiment, the JB and FLJ populations were passed through common rearing conditions for one generation to eliminate non\u0026ndash;genetic parental effects. Eggs were collected in 40 vials per population at a density of ~50 eggs in 6mL SM in each vial and incubated at SLC. All the flies that eclosed from these vials at the end of the 10\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday for FLJ and on the 12\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday for JB from the ECD were transferred to plexiglass cages with sterile SM food plates. These flies were referred to as standardized flies. The assay flies were generated from the standardized flies and egg collection was staggered by the developmental time difference to obtain assay flies of similar-age for experiments\u003csup\u003e22,24\u003c/sup\u003e. After red eyes became visible in pupae, vigil checks were carried-out at every 4 hour interval. Emerged flies were sorted according to their sex and maintained in unisex vials with 6 mL SM till used in other experiments. Flies were sorted until 80% of the flies had eclosed. \u0026nbsp;The virgin flies of a given gender were pooled before being used in various assays.\u003c/p\u003e\n\u003cp\u003eThe purpose of this study was to assess the \u0026lsquo;live fast, die young\u0026rsquo; hypothesis in males. Hence, in order to minimize the influence of the females we have used the females from the respective JB population in all the assays. Two types of mating pairs were set up: JB female \u0026times; FLJ male and JB female \u0026times; JB male.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of Energy reserves at emergence:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive samples of 10 virgin male flies per replicate population of each selection type were prepared for estimation of carbohydrates, proteins and lipids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1) Carbohydrates estimation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal carbohydrates was assayed using Anthrone method\u003csup\u003e25\u003c/sup\u003e with slight modifications. Virgin flies were homogenized in 200\u0026mu;l of 0.1M PBS. Samples were centrifuged at 12,000 rpm for 10 minutes at 4 \u0026deg;C. 5x dilution was prepared by adding Milli-Q water to samples. 800\u0026mu;l of anthrone reagent (Merck- CAS No. 90-44-8) was added, and the reaction mixture was boiled at 90 \u0026deg;C in a water bath for 18-20 minutes. Samples were cooled to room temperature and were loaded into the 96-well plate. Each biological sample was plated in triplicate. Absorbance was measured at 625nm using ELISA plate reader. The standard glucose curve was used to estimate total carbohydrates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2) Protein estimation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein estimation was performed using Pierce\u003csup\u003etm\u0026nbsp;\u003c/sup\u003eBCA Protein Assay Kit (Thermo Scientific, Catalog no.-23227) following manufacturer protocol. Virgin flies were homogenized in 300\u0026mu;l 0.1M PBS. To make the working reagent, 50 parts of BCA reagent A with 1 Part of BCA reagent B were mixed (50 BCA reagent A: 1 BCA reagent B). Standard and samples were plated on 96-well plates and 200\u0026mu;l of working reagent was added to both. Each biological sample was plated in triplicate. The plate was agitated on a plate shaker for 30 seconds and incubated for 30 minutes at 30 \u0026deg;C. Absorbance was taken at 562nm by using ELISA plate reader. Bovine serum albumin was used to make a standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3) Lipid estimation: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVirgin flies were used to obtain the fresh weight of the flies using microbalance (Model No. CM11, Citizen). Samples were stored in glass vials and dried in hot air oven at 70 \u0026deg;C for 36 hours and weighed again to obtain the dry weight. Flies were transferred to pre-labelled 1.5 ml micro centrifuge tubes. Diethyl ether was used to extract ether-soluble lipids by following the protocol of Handa et. al., 2014 with few modifications\u003csup\u003e16\u003c/sup\u003e . Lipids were extracted for over a duration for 48 hours with three ether changes with 12 hours interval. After the last ether change, flies oven dried at 47 \u0026deg;C for ~2 hours and weight was again reassed to obtain lipid-free weight of the flies. The difference between lipid-free weight and dry weight of the flies was used to calculate lipid content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnergy estimation:\u0026nbsp;\u003c/strong\u003eThe carbohydrate, protein and lipid data were multiplied by their calorific values 4.2 cal/mg, 4.19 cal/mg and 9.5 cal/mg respectively\u003csup\u003e26\u003c/sup\u003e. The resulting data was summed and averaged per replicate population in order to obtain the total energy levels of flies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReproduction related traits assay:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1) Pre-copulatory traits:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVirgin male and female flies were aged for 3 days before pairing them for assessment of pre-copulatory behavioural traits- total courtship duration (sec) and number of mounting attempts, and copulation duration (sec). Each male-female pair was introduced into mating chamber of 1.7 cm height \u0026times; 2 cm diameter dimension. All activities performed by the pair was video recorded till the disengagement post copulating pair or for a maximum duration of 1 hr in case of pairs that did not copulate\u003csup\u003e27\u003c/sup\u003e. Pre-copulatory behaviour data were extracted by viewing video recordings. Pre-copulatory traits and copulation duration were assessed on only one replicate population each of FLJ and JB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2) Life-time fecundity, fecundity profile and realized fitness traits:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndividual male-and female pair were transferred to sterile 3mL SM vials. Thirty such pairs per treatment per replicate population were set-up and held in Powers Scientific Incubators USA, environmental chambers. The pairs were flipped into fresh SM vials every 24 hours. Eggs laid in the preceding 24 hour period were counted under Zeiss Stemi DV4 stereo zoom microscope. This process was carried out till the death of all flies. The total life-time fecundity and fecundity profile were obtained from this data. In order to assess the realized fitness, we incubated egg vials at pre-determined adult ages and counted the number of emerging adults to estimate the % viability. We incubated 15 vials containing maximum number of eggs during the first half of the experimental duration and nearly all vials during the latter half the experimental.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLongevity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the mortality data from the reproduction related traits assay set-up, we constructed Kaplan-Meir survival probability curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the traits data was subjected to Shapiro-Wilk normality test. All data that passed the normality test, were subjected to Bartlett\u0026rsquo;s test of homogeneity\u003csup\u003e13\u003c/sup\u003e. The energy equivalents and life-time fecundity passed the normality test and hence we adopted one-way ANOVA model with selection as a fixed factor and replicate block as a random factor. The pre-copulatory traits data failed the normality test, hence were subjected to non-parametric, Mann Whitney U test. Male fertility data passed the normality test, hence was subjected to two-way ANOVA with selection and days as fixed factors, and replicate blocks as random factor. The survival probabilities of males were assessed using non-parametric Kaplan Meier analysis followed by log-rank (Mantel-Cox) test using Graphpad version 8.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Institute of Eminence (IOE), University of Delhi grant (Ref. No./IOE/2023-24/12/FRP) to MS. We thank Council for Scientific \u0026amp; Industrial Research (CSIR) for research fellowship to AKF (File No. 09/0045(12766)/2021-EMR-1). We also thank University Grants Commission (UGC) for research fellowship to AY (Ref. No. 211610167457) and to NKS (Ref. No. 19/06/2016(i)EU-V-346377).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AKF, MS\u003c/p\u003e\n\u003cp\u003eMethodology, investigation, validation: AKF, NKS, NR, MK, AY\u003c/p\u003e\n\u003cp\u003eFunding acquisition and supervision: MS\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: AKF\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: AKF, NKS, MS\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available as supplementary sheets.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKirkwood, T. B. L. Evolution of ageing. 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Effect of Heavy Metal Tolerance Induced Oxidative Stress on Energy Metabolism in \u0026lt;\u0026thinsp;em\u0026thinsp;\u0026gt;\u0026thinsp;Drosophila melanogaster\u0026lt;/em\u0026gt;. \u003cem\u003eExpert Opinion on Environmental Biology\u003c/em\u003e 2018, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVon Philipsborn, A. C., Shohat-Ophir, G. \u0026amp; Rezaval, C. Single-Pair Courtship and Competition Assays in \u003cem\u003eDrosophila\u003c/em\u003e. \u003cem\u003eCold Spring Harb Protoc\u003c/em\u003e 2023, pdb.prot108105 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4516780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4516780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn holometabolus insects, fitness is intricately linked with body size. Large males with high energy reserves often adopt \u0026lsquo;live fast, die young\u0026rsquo; life-history strategy to maximize fitness compared to small individuals. However, in promiscuous, iteroparous systems, males that live fast and die late have evolutionary advantage. Using two types of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e populations- one that have evolved small size as a response to long term selection for faster development and extended longevity (FLJs) and the other that are large ancestral control populations (JBs), we revisit the (i) disposable soma theory, and (ii) live fast, die early life history strategy hypothesis. Contrary to the two hypotheses, the small FLJ males with significantly reduced energy reserves invested heavily in pre-reproductive traits as indicated by significantly higher courtship duration, number of mounting attempts, and comparable copulation duration to JB males. FLJ males also have comparable lifetime fecundity and realized fitness without compromising on longevity, suggesting that FLJ males are adopting a novel \u0026ldquo;live fast, die late\u0026rdquo; life history strategy.\u003c/p\u003e","manuscriptTitle":"Evolution of “Live fast, die late” life history strategy in Drosophila melanogaster males","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 15:14:33","doi":"10.21203/rs.3.rs-4516780/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb249dda-3e73-4edc-acdc-2219a5750e7c","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32910624,"name":"Biological sciences/Evolution/Experimental evolution"},{"id":32910625,"name":"Biological sciences/Evolution/Evolutionary theory"}],"tags":[],"updatedAt":"2024-06-20T15:14:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 15:14:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4516780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4516780","identity":"rs-4516780","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}