Idiosyncratic effects of bacterial infection on female fecundity in Drosophila melanogaster.

Existing theories make different predictions regarding the effect of a pathogenic infection on the host capacity to reproduce. Terminal investment theory suggests that due to the increased risk of mortality, and the associated risk of losing future opportunity to reproduce, infected individuals would increase their investment towards reproduction. Life-history theory posits that due to energetic and resource costs associated with mounting an immune defense, hosts would decrease their investment towards reproduction, and reallocate resources towards defense and survival. Additionally, Somatic damage incurred by the host due to the infection is also expected to compromise the host capacity to reproduce. We explored these possibilities in Drosophila melanogaster females experimentally infected with pathogenic bacteria. We tested if the effect of infection on female fecundity is pathogen specific, determined by infection outcome, and variable between individual infected females. We observed that the mean, population level change in post-infection female fecundity was pathogen specific, but not correlated with mortality risk. Furthermore, infection outcome, i.e., if the infected female died or survived the infection, had no effect on fecundity at this level. At individual resolution, females that died after infection exhibited greater variation in fecundity compared to ones that survived the infection. This increased variation was bidirectional, with some females reproducing in excess while others reproducing less compared to the controls. Altogether, our results suggest that post-infection female fecundity is unlikely to be driven by risk of mortality and is probably determined by the precise physiological changes that an infected female undergoes when infected by a specific pathogen.

Starvation increases susceptibility to bacterial infection and promotes systemic pathogen proliferation in Drosophila melanogaster females.

Defense against pathogens and parasites requires substantial investment of energy and resources on part of the host. This makes the host immune function dependent on availability and accessibility of resources. A resource deprived host is therefore expected to be more susceptible to infections, although empirical results do not always align with this prediction. Limiting host access to resources can additionally impact within-host pathogen numbers, either directly by altering the amount of resources available to the pathogens for proliferation or indirectly by altering the efficiency of the host immune system. We tested for the effects of host starvation (complete deprivation of resources) on susceptibility to bacterial pathogens, and within-host pathogen proliferation, in Drosophila melanogaster females. Our results show that starvation increases post-infection mortality of the host, but in a pathogen-specific manner. This increase in mortality is always accompanied by increased within-host pathogen proliferation. We therefore propose that starvation compromises host resistance to bacterial infections in Drosophila melanogaster females thereby increasing susceptibility to infections.

Experimental adaptation to pathogenic infection ameliorates negative effects of mating on host post-infection survival in Drosophila melanogaster.

Sexual activity (mating) negatively affects immune function in various insect species across both sexes. In Drosophila melanogaster females, mating increases susceptibility to pathogenic challenges and encourages within-host pathogen proliferation. This effect is pathogen and host genotype dependent. We tested if mating-induced increased susceptibility to infections is more, or less, severe in hosts experimentally adapted to pathogenic infection. We selected replicate D. melanogaster populations for increased post-infection survival following infection with a bacterial pathogen, Enterococcus faecalis. We found that females from the selected populations were better at surviving a pathogenic infection compared to the females from the control populations. This was true in the case of both the pathogen used for selection and other novel pathogens (i.e., pathogens the hosts have not encountered in recent history). Additionally, the negative effect of mating on post-infection survival was limited to only the females from control populations. Therefore, we have demonstrated that experimental selection for increased post-infection survival ameliorates negative effects of mating on host susceptibility to infections.

Experimental evolution for improved post-infection survival selects for increased disease resistance in Drosophila melanogaster.

Disease resistance (defined as the host capacity to limit systemic infection intensity) and disease tolerance (defined as the host capacity to limit infection-induced damage) are two complementary defense strategies that help the hosts maximize their survival and fitness when infected with pathogens and parasites. In addition to the underlying physiological mechanisms, existing theory postulates that these two strategies differ in terms of the conditions under which each strategy evolves in the host populations, their evolutionary dynamics, and the ecological and epidemiological consequences of their evolution. Here we explored if one or both of these strategies evolve when host populations are subjected to selection for increased post-infection survival. We experimentally evolved Drosophila melanogaster populations, selecting for the flies that survived an infection with the entomopathogen Enterococcus faecalis. We found that the host populations evolved increased disease resistance in response to selection for increased survival. This was despite the physiological costs associated with increased resistance, expression of which varied with the phase of infection. We did not find evidence of any change in disease tolerance in the evolved host populations.

Consequences of adaptation to larval crowding on sexual and fecundity selection in Drosophila melanogaster.

Sexual selection is a major force influencing the evolution of sexually reproducing species. Environmental factors such as larval density can manipulate adult condition and influence the direction and strength of sexual selection. While most studies on the influence of larval crowding on sexual selection are either correlational or single-generation manipulations, it is unclear how evolution under chronic larval crowding affects sexual selection. To answer this, we measured the strength of sexual selection on male and female Drosophila melanogaster that had evolved under chronic larval crowding for over 250 generations in the laboratory, along with their controls which had never experienced crowding, in a common garden high-density environment. We measured selection coefficients on male mating success and sex-specific reproductive success, as separate estimates allowed dissection of sex-specific effects. We show that experimental evolution under chronic larval crowding decreases the strength of sexual and fecundity selection in males but not in females, relative to populations experiencing crowding for the first time. The effect of larval crowding in reducing reproductive success is almost twice in females than in males. Our study highlights the importance of studying how evolution in a novel, stressful environment can shape adult fitness in organisms.

Pten associates with important gene regulatory network to fine-tune Müller glia-mediated zebrafish retina regeneration.

Unlike mammals, zebrafish possess a remarkable ability to regenerate damaged retina after an acute injury. Retina regeneration in zebrafish involves the induction of Müller glia-derived progenitor cells (MGPCs) exhibiting stem cell-like characteristics, which are capable of restoring all retinal cell-types. The induction of MGPC through Müller glia-reprograming involves several cellular, genetic and biochemical events soon after a retinal injury. Despite the knowledge on the importance of Phosphatase and tensin homolog (Pten), which is a dual-specificity phosphatase and tumor suppressor in the maintaining of cellular homeostasis, its importance during retina regeneration remains unknown. Here, we explored the importance of Pten during zebrafish retina regeneration. The Pten gets downregulated upon retinal injury and is absent from the MGPCs, which is essential to trigger Akt-mediated cellular proliferation essential for retina regeneration. We found that the downregulation of Pten in the post-injury retina accelerates MGPCs formation, while its overexpression restricts the regenerative response. We observed that Pten regulates the proliferation of MGPCs not only through Akt pathway but also by Mmp9/Notch signaling. Mmp9-activity is essential to induce the proliferation of MGPCs in the absence of Pten. Lastly, we show that expression of Pten is fine-tuned through Mycb/histone deacetylase1 and Tgf-ß signaling. The present study emphasizes on the stringent regulation of Pten and its crucial involvement during the zebrafish retina regeneration.

Evolution of cross-tolerance in Drosophila melanogaster as a result of increased resistance to cold stress.

Cold stress is a critical environmental challenge that affects an organism's fitness-related traits. In Drosophila, increased resistance to specific environmental stress may lead to increased resistance to other kinds of stress. In the present study, we aimed to understand whether increased cold stress resistance in Drosophila melanogaster can facilitate their ability to tolerate other environmental stresses. For the current study, we used successfully selected replicate populations of D. melanogaster against cold shock and their control population. These selected populations have evolved several reproductive traits, including increased egg viability, mating frequency, male mating ability, ability to sire progenies, and faster recovery for mating latency under cold shock conditions. In the present work, we investigated egg viability and mating frequency with and without heat and cold shock conditions in the selected and their control populations. We also examined resistance to cold shock, heat shock, desiccation, starvation, and survival post-challenge with Staphylococcus succinus subsp. succinus PK-1 in the selected and their control populations. After cold-shock treatment, we found a 1.25 times increase in egg viability and a 1.57 times increase in mating frequency in the selected populations compared to control populations. Moreover, more males (0.87 times) and females (1.66 times) of the selected populations survived under cold shock conditions relative to their controls. After being subjected to heat shock, the selected population's egg viability and mating frequency increased by 0.30 times and 0.57 times, respectively, compared to control populations. Additionally, more selected males (0.31 times) and females (0.98 times) survived under heat shock conditions compared to the control populations. Desiccation resistance slightly increased in the females of the selected populations relative to their control, but we observed no change in the case of males. Starvation resistance decreased in males and females of the selected populations compared to their controls. Our findings suggest that the increased resistance to cold shock correlates with increased tolerance to heat stress, but this evolved resistance comes at a cost, with decreased tolerance to starvation.

Investment in adult reproductive tissues is affected by larval growth conditions but not by evolution under poor larval growth conditions in Drosophila melanogaster.

In many insects, the larval environment is confined to the egg-laying site, which often leads to crowded larval conditions, exposing the developing larvae to poor resource availability and toxic metabolic wastes. Larval crowding imposes two opposing selection pressures. On one hand, due to poor nutritional resources during developmental stages, adults from the crowded larval environment have reduced investment in reproductive tissues. On the other hand, a crowded larval environment acts as a cue for future reproductive competition inducing increased investment in reproductive tissues. Both these selection pressures are likely affected by the level of crowding. The evolutionary consequence of adaptation to larval crowding environment on adult reproductive investment is bound to be a result of the interaction of these two opposing forces. In this study, we used experimentally evolved populations of Drosophila melanogaster adapted to larval crowding to investigate the effect of adaptation to larval crowding on investment in reproductive organs (testes and accessory glands) of males. Our results show that there is a strong effect of larval developmental environment on absolute sizes of testes and accessory glands. However, there was no effect of the developmental environment when testis size was scaled by body size. We also found that flies from crowded cultures had smaller accessory gland sizes relative to body size. Moreover, the sizes of the reproductive organs were not affected by the selection histories of the populations. This study highlights that adaptation to two extremely different developmental environments does not affect the patterns of reproductive investment. We discuss the possibility that differential investment in reproductive tissues could be influenced by the mating dynamics and/or investment in larval survival traits, rather than just the developmental environment of the populations.

Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation.

BACKGROUND: In spatially structured populations, local adaptation improves organisms' fitness in their native environment. Hosts and pathogens can rapidly adapt to their local antagonist. Since males and females can differ in their immunocompetence, the patterns of local adaptation can be different between the sexes. However, there is little information about sex differences in local adaptation in host-pathogen systems. RESULTS: In the current study, we experimentally coevolved four different replicate populations of Drosophila melanogaster (host) and Pseudomonas entomophila (pathogen) along with appropriate controls. We used the four host-pathogen coevolution populations to investigate the occurrence of local adaptation separately in males and females of the coevolving hosts. We also assessed local adaptation in pathogens. We set up a reciprocal infection experiment where we infected each of the four coevolving hosts with their local pathogen or non-local pathogens from the other three replicate populations. We found that overall, male and female hosts had better survivorship when infected with local pathogens, indicating that they were locally adapted. Interestingly, males were more susceptible to non-local pathogens compared to females. In addition, we found no fecundity cost in females infected with either local or non-local pathogens. We found no evidence of local adaptation among the pathogens. CONCLUSION: Our study showed sex-specific adaptation in the coevolving hosts where female hosts had a broader response against allopatric coevolving pathogens with no cost in fecundity. Thus, our results might suggest a novel mechanism that can maintain variation in susceptibility in spatially structured populations.

Male mating success evolves in response to increased levels of male-male competition.

Male-biased operational sex ratios can increase male-male competition and can potentially select for both increased pre- and postcopulatory male success. In the present study, using populations of Drosophila melanogaster evolved under male-biased (M) or female-biased (F) sex ratios, we asked whether (a) male mating success can evolve, (b) males are better at mating females that they have coevolved with, (c) males mating success is affected by female mating status, and (d) male mating success is correlated with their courtship effort. We directly competed M and F males for mating with (a) virgin ancestral (common) females, (b) virgin females from the M and F populations, and (c) singly mated females from the M and F populations. We also assessed the courtship frequency of the males when paired with mated M or F females. Our results show that M males, evolving under an increased level of male-male competition, have higher mating success than F males irrespective of the female evolutionary history. However, the difference in mating success is more pronounced if the females had mated before. M males also have a higher courtship frequency than F males, but we did not find any correlation between mating success and courtship frequency.

No major cost of evolved survivorship in Drosophila melanogaster populations coevolving with Pseudomonas entomophila.

Rapid exaggeration of host and pathogen traits via arms race dynamics is one possible outcome of host-pathogen coevolution. However, the exaggerated traits are expected to incur costs in terms of resource investment in other life-history traits. The current study investigated the costs associated with evolved traits in a host-pathogen coevolution system. We used the Drosophila melanogaster (host)-Pseudomonas entomophila (pathogen) system to experimentally derive two selection regimes, one where the host and pathogen both coevolved, and the other, where only the host evolved against a non-evolving pathogen. After 17 generations of selection, we found that hosts from both selected populations had better post-infection survivorship than controls. Even though the coevolving populations tended to have better survivorship post-infection, we found no clear evidence that the two selection regimes were significantly different from each other. There was weak evidence for the coevolving pathogens being more virulent than the ancestral pathogen. We found no major cost of increased post-infection survivorship. The costs were not different between the coevolving hosts and the hosts evolving against a non-evolving pathogen. We found no evolved costs in the coevolving pathogens. Thus, our results suggest that increased host immunity and pathogen virulence may not be costly.

Investigating the interaction between inter-locus and intra-locus sexual conflict using hemiclonal analysis in Drosophila melanogaster.

BACKGROUND: Divergence in the evolutionary interests of males and females leads to sexual conflict. Traditionally, sexual conflict has been classified into two types: inter-locus sexual conflict (IeSC) and intra-locus sexual conflict (IaSC). IeSC is modeled as a conflict over outcomes of intersexual reproductive interactions mediated by loci that are sex-limited in their effects. IaSC is thought to be a product of selection acting in opposite directions in males and females on traits with a common underlying genetic basis. While in their canonical formalisms IaSC and IeSC are mutually exclusive, there is growing support for the idea that the two may interact. Empirical evidence for such interactions, however, is limited. RESULTS: Here, we investigated the interaction between IeSC and IaSC in Drosophila melanogaster. Using hemiclonal analysis, we sampled 39 hemigenomes from a laboratory-adapted population of D. melanogaster. We measured the contribution of each hemigenome to adult male and female fitness at three different intensities of IeSC, obtained by varying the operational sex ratio. Subsequently, we estimated the intensity of IaSC at each sex ratio by calculating the intersexual genetic correlation (rw,g,mf) for fitness and the proportion of sexually antagonistic fitness-variation. We found that the intersexual genetic correlation for fitness was positive at all three sex ratios. Additionally, at male biased and equal sex ratios the rw,g,mf was higher, and the proportion of sexually antagonistic fitness variation lower, relative to the female biased sex ratio, although this trend was not statistically significant. CONCLUSION: Our results indicate a statistically non-significant trend suggesting that increasing the strength of IeSC ameliorates IaSC in the population.

Experimental evolution reveals sex-specific dominance for surviving bacterial infection in laboratory populations of Drosophila melanogaster.

Males and females are subjected to distinct kinds of selection pressures, often leading to the evolution of sex-specific genetic architecture, an example being sex-specific dominance. Sex-specific dominance reversals (SSDRs), where alleles at sexually antagonistic loci are at least partially dominant in the sex they benefit, have been documented in Atlantic salmon, rainbow trout, and seed beetles. Another interesting feature of many sexually reproducing organisms is the asymmetric inheritance pattern of X chromosomes, which often leads to distinct evolutionary outcomes on X chromosomes compared to autosomes. Examples include the higher efficacy of sexually concordant selection on X chromosomes, and X chromosomes being more conducive to the maintenance of sexually antagonistic polymorphisms under certain conditions. Immunocompetence is a trait that has been extensively investigated for sexual dimorphism with growing evidence for sex-specific or sexually antagonistic variation. X chromosomes have been shown to harbor substantial immunity-related genetic variation in the fruit fly, Drosophila melanogaster. Here, using interpopulation crosses and cytogenetic cloning, we investigated sex-specific dominance and the role of the X chromosome in improved postinfection survivorship of laboratory populations of D. melanogaster selected against pathogenic challenge by Pseudomonas entomophila. We could not detect any contribution of the X chromosome to the evolved immunocompetence of our selected populations, as well as to within-population variation in immunocompetence. However, we found strong evidence of sex-specific dominance related to surviving bacterial infection. Our results indicate that alleles that confer a survival advantage to the selected populations are, on average, partially dominant in females but partially recessive in males. This could also imply an SSDR for overall fitness, given the putative evidence for sexually antagonistic selection affecting immunocompetence in Drosophila melanogaster. We also highlight sex-specific dominance as a potential mechanism of sex differences in immunocompetence, with population-level sex differences primarily driven by sex differences in heterozygotes.

Evolution of reproductive traits have no apparent life-history associated cost in populations of Drosophila melanogaster selected for cold shock resistance.

BACKGROUND: In insect species like Drosophila melanogaster, evolution of increased resistance or evolution of particular traits under specific environmental conditions can lead to energy trade-offs with other crucial life-history traits. Adaptation to cold stress can, in principle, involve modification of reproductive traits and physiological responses. Reproductive traits carry a substantial cost; and therefore, the evolution of reproductive traits in response to cold stress could potentially lead to trade-offs with other life-history traits. We have successfully selected replicate populations of Drosophila melanogaster for increased resistance to cold shock for over 33 generations. In these populations, the ability to recover from cold shock, mate, and lay fertile eggs 24 h post cold shock is under selection. These populations have evolved a suite of reproductive traits including increased egg viability, male mating ability, and siring ability post cold shock. These populations also show elevated mating rate both with and without cold shock. In the present study, we quantified a suite of life-history related traits in these populations to assess if evolution of cold shock resistance in these populations comes at a cost of other life-history traits. RESULTS: To assess life-history cost, we measured egg viability, mating frequency, longevity, lifetime fecundity, adult mortality, larva to adult development time, larvae to adults survival, and body weight in the cold shock selected populations and their controls under two treatments (a) post cold chock and (b) without cold shock. Twenty-four hours post cold shock, the selected population had significantly higher egg viability and mating frequency compared to control populations indicating that they have higher cold shock resistance. Selected populations had significantly longer pre-adult development time compared to their control populations. Females from the selected populations had higher body weight compared to their control populations. However, we did not find any significant difference between the selected and control populations in longevity, lifetime fecundity, adult mortality, larvae to adults survival, and male body weight under the cold chock or no cold shock treatments. CONCLUSIONS: These findings suggest that cold shock selected populations have evolved higher mating frequency and egg viability. However, there is no apparent life-history associated cost with the evolution of egg viability and reproductive performances under the cold stress condition.

Enemies make you stronger: Coevolution between fruit fly host and bacterial pathogen increases postinfection survivorship in the host.

Multiple laboratory studies have evolved hosts against a nonevolving pathogen to address questions about evolution of immune responses. However, an ecologically more relevant scenario is one where hosts and pathogens can coevolve. Such coevolution between the antagonists, depending on the mutual selection pressure and additive variance in the respective populations, can potentially lead to a different pattern of evolution in the hosts compared to a situation where the host evolves against a nonevolving pathogen. In the present study, we used Drosophila melanogaster as the host and Pseudomonas entomophila as the pathogen. We let the host populations either evolve against a nonevolving pathogen or coevolve with the same pathogen. We found that the coevolving hosts on average evolved higher survivorship against the coevolving pathogen and ancestral (nonevolving) pathogen relative to the hosts evolving against a nonevolving pathogen. The coevolving pathogens evolved greater ability to induce host mortality even in nonlocal (novel) hosts compared to infection by an ancestral (nonevolving) pathogen. Thus, our results clearly show that the evolved traits in the host and the pathogen under coevolution can be different from one-sided adaptation. In addition, our results also show that the coevolving host-pathogen interactions can involve certain general mechanisms in the pathogen, leading to increased mortality induction in nonlocal or novel hosts.

Evolution of sex-specific heat stress tolerance and larval Hsp70 expression in populations of Drosophila melanogaster adapted to larval crowding.

The ability to tolerate temperature stress is an important component of adult fitness. In holometabolous insects like Drosophila melanogaster, adult stress resistance can be affected by growth conditions experienced during the larval stages. Although evolution under crowded larval conditions is known to lead to the correlated evolution of many adult traits, its consequences on adult heat stress tolerance have not been investigated. Therefore, in the present study, we assessed the adult heat stress tolerance in populations of D. melanogaster adapted to a stressful larval crowding environment. We used replicate populations of D. melanogaster, selected for adaptation to larval crowding stress (MCUs), for more than 230 generations, and their respective controls (MBs). Larvae from selected and control populations were grown under crowded and uncrowded conditions, and their adult heat shock resistance at two different temperatures was measured. Further, we compared Hsp70 expression in crowded and uncrowded larvae of both populations and also measured the Hsp70 expression after a mild heat treatment in adults of selected and control populations. Our results showed that adaptation to larval crowding leads to the evolution of Hsp70 gene expression in larval stages and improves adult heat stress tolerance ability in males, but not in females.

Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex ratios.

Post-copulatory sexual selection (PSS) is an important selective force that determines fitness in polyandrous species. PSS can be intense in some cases and can drive the evolution of remarkable ejaculate properties. In males, investment in ejaculate plays an important role in the outcome of PSS. Thus, males are expected to adaptively tailor their ejaculate according to the perceived competition in their vicinity. Plastic responses in ejaculate investment to variation in intrasexual competition are disparate and widespread in males. We investigated the evolution of plasticity in reproductive traits using Drosophila melanogaster populations evolving for more than 150 generations under male- or female-biased sex ratios. When exposed to different numbers of competitors early in their life, males from these two regimes responded differently in terms of their copulation duration and sperm competitive ability. In addition, the effect of this early life experience wore off at different rates in males of male-biased and female-biased regimes with increasing time from the removal of competitive cues. Furthermore, our study finds that males change their reproductive strategies depending upon the identity of rival males. Together, our results provide evidence of the evolution of male reproductive investment that depends on socio-sexual cues experienced early in life.

Absence of reproduction-immunity trade-off in male Drosophila melanogaster evolving under differential sexual selection.

BACKGROUND: The theory of trade-off suggests that limited resources should lead to trade-off in resource intensive traits such as, immunity related and sexually selected traits in males. Alternatively, sexual exaggerations can also act as an honest indicator of underlying immunocompetence, leading to positive correlations between these traits. Evidences in support of either hypothesis in invertebrates are equivocal. Whereas several studies have addressed this question, few have used naturally occurring pathogens and realized post infection realized immunity (e.g., survivorship) to assay the fitness correlations between these two sets of traits. RESULTS: Adopting an experimental evolution approach, we evolved replicate populations of Drosophila melanogaster under high and low sexual selection regimes for over a hundred generations and found the following in virgin and mated males in three separate assays: a.Post infection survivorship against two natural pathogens - Pseudomonas entomophila (Pe) and Staphylococcus succinus (Ss): Mated males survived better against Pe, but were no different than virgins against Ss.b.Bacterial clearance ability against a third natural pathogen Providencia rettgeri (Pr): Mated males had significantly lower CFUs than virgins. However, sexual selection history had no effect on realized immunity of either virgin or mated males. CONCLUSION: We show that while mating can affect realized immunity in a pathogen specific way, sexual selection did not affect the same. The results highlight that complex polygenic traits such as immunity and reproductive traits not necessarily evolve following a binary trade-off model. We also stress the importance natural pathogens while studying sexual selection-immunity correlations.

Reproductive Isolation through Experimental Manipulation of Sexually Antagonistic Coevolution in Drosophila melanogaster.

Promiscuity can drive the evolution of sexual conflict before and after mating occurs. Post mating, the male ejaculate can selfishly manipulate female physiology, leading to a chemical arms race between the sexes. Theory suggests that drift and sexually antagonistic coevolution can cause allopatric populations to evolve different chemical interactions between the sexes, thereby leading to postmating reproductive barriers and speciation. There is, however, little empirical evidence supporting this form of speciation. We tested this theory by creating an experimental evolutionary model of Drosophila melanogaster populations undergoing different levels of interlocus sexual conflict. We found that allopatric populations under elevated sexual conflict show assortative mating, indicating premating reproductive isolation. Further, these allopatric populations also show reduced copulation duration and sperm defense ability when mating happens between individuals across populations compared to that within the same population, indicating postmating prezygotic isolation. Sexual conflict can cause reproductive isolation in allopatric populations through the coevolution of chemical (postmating prezygotic) as well as behavioural (premating) interactions between the sexes. Thus, to our knowledge, we provide the first comprehensive evidence of postmating (as well as premating) reproductive isolation due to sexual conflict.

Virility does not imply immensity: Testis size, accessory gland size and ejaculate depletion pattern do not evolve in response to experimental manipulation of sex ratio in Drosophila melanogaster.

Sperm competition theory predicts that with increase in sperm competition, males either invest more in reproductive organ(s) and/or improve ejaculate investment. We test this idea using experimental evolution in Drosophila melanogaster. We maintained replicate populations of Drosophila melanogaster under male (M) and female (F) biased sex ratio regimes for more than a hundred generations with the result that males from the M regime evolved higher sperm competitive abilities relative to males from the F regime. In the present study, we measured the testes and the accessory gland size of virgin and singly mated males from the M and F regimes. The M and F males do not differ in either testis or accessory gland size. Additionally, ejaculate investment is not different in the M and F males, as measured by reduction in testis and accessory gland sizes. Thus, contrary to theoretical prediction and evidence from other species, we found that evolved differences in sperm competitive ability are not necessarily due to evolution of testis/accessory gland size or strategic ejaculate investment in these populations.