Drosophila
By Aaron King and Nicholas Gentry, PhD
Expert review provided by Zita Santos, PhD
Key Terminology
- Eclosion - emergence as an adult from the pupae or as a larva from the egg
- Diapause - a delay in maturation in response to environmental conditions such as poor nutrition, suboptimal temperature, and others. Development of sexual maturity pauses temporarily.
- Sensitive Period - portion of lifespan where Drosophila is able to induce diapause. Once the Sensitive Period has passed, the program cannot be altered.
- Fertility - Actual production of offspring. In this case, fertilized egg production and subsequent health of the offspring.
- Fecundity - Ability to produce offspring. In this case, oocyte production.
Key Reproductive Longevity Stats for Drosophilia
- Up to 100 eggs can be laid per day on a nutrient rich diet [Drummond-Barbosa and Spradling, 2001] but more commonly lay up to 60 per day [Klepsatel et al, 2013; Miller et al, 2014]. Peak egg laying occurs around 4-5 days after eclosion, with infertility occurring around 35-50 days post-eclosion, depending on the strain and study. [Zhao et al, 2008; Klepsatel et al, 2013; Miller et al, 2014]. There is conflicting evidence whether there is a steep drop off in fertility around 35-40 days of age or a gradual decline to zero [Zhao et al, 2008; Klepsatel et al, 2013]. (Figure 1).
- Mating is an important factor regulating the activation and progression of oogenesis via the action of Sex peptide provided by the seminal fluid. Virgin females lay a residual number of last stage oocytes, a process which is increased dramatically upon mating, independently of the age that mating occurred [Kubli, 2003; Greenblatt et al, 2019].
- Lifespan of lab-grown strains of Drosophila have a median lifespan of approximately 70 days and a maximum around 90 days [Ziehm et al, 2013]
- Eclosion occurs 10-11 days after hatching from an egg [An Introduction to Drosophila Melanogaster].
- It is important to note that ovipository period differs from the true fertility period. That is, females may still lay inviable eggs for 5-10 days beyond the period when these eggs actually hatch [Klepsatel et al, 2013]. Hatching rates must be carefully monitored and distinguished from egg-laying to accurately measure fertility. (Figure 2)
- Both males and females are fertile and sexually mature within 24 hours after eclosion and usually mate within 8-10 hours after this event [An Introduction to Drosophila Melanogaster; Stromnaes and Oistein, 1959]
- The optimal living conditions for drosophila melanogaster is 25°C at 60% humidity and 12:12 Light:Dark conditions [An Introduction to Drosophila Melanogaster; Schwartz et al, 1985] . Temperatures below 18C or altered photoperiods can increase time to sexual maturity and reproductive cycling [Saunders et al, 1989].
Advantages
+ Fast Turnaround time: Drosophila have a much faster life cycle than mice or humans (~ 3 months), so there is less time required per generation to measure reproductive lifespan.
+ Well Developed Visualization Tools: Fluorescent markers have been hybridized to many fly genes of interest to better visualize activity under the microscope. Live imaging of the ovarian stem cell niche was recently refined [Wilcockson and Ashe, 2021]. Antibody staining is frequently used for visualizing membrane structures and has been effective for decades. Genetic-based visualization tools are also broadly available.
+ In Vitro Models of Germ Cells: Unlike most other model organisms, Drosophila has stable and well developed in vitro models of ovaries [Niki et al, 2006].
+ Easy to Genetically Modify: The 4 chromosomes of flies contain a surprisingly similar number of active genes compared to humans. The reduced chromosome count and gene redundancy makes it much easier to modify fly genetics to identify specific gene function [Armstrong, 2020]. The fly’s relative genetic similarity to humans makes this tool especially powerful. Similar to overall lifespan, it is possible to genetically alter flies to change their age of reproductive senescence [Miller et al, 2014].
+ Conserved nutrient response pathways: Many key components of mammalian nutrient response pathways have Drosophila orthologs (TOR pathway, GCN2 pathway, etc.)
+ Stable Cell Lines: Stable lines of Drosophila germline stem cells have been maintained for at least a decade [Niki et al, 2006].
+ Large community of support:
Many links to online resources, a few of which are listed further below.
+Post-reproductive period: Drosophila females live for a substantial (~30-40%) portion of their lifespan after the cessation of fertility, which can be useful for the study of reproductive longevity intervention [Miller et al, 2014].
Disadvantages
- No Menopause in Drosophila: y. Despite a relatively lengthy post-reproductive lifespan, Drosophila, like most animals [Ellis et al, 2018; Croft et al, 2015], do not experience an extended biologically planned post-reproductive period akin to menopause; instead, the reproductive system declines in tandem with the rest of the organism. Reported fly (and other animal) post-reproductive lifespans may also be driven by laboratory conditions, further complicating direct comparison. The viability of direct experimental translatability to human menopause is therefore complex, and findings in this model should be interpreted with caution.
- Oogenesis: Unlike humans who are born with all their eggs, oogenesis occurs in drosophila females for most of their adult life. Drosophila have Germline Stem Cells, but humans may not [Wong et al, 2005]. While germline stem cells have been reported in humans [Cheng et al, 2022], the findings remain controversial and unvalidated. The presence of GSCs makes the fundamental regenerative and aging biology of these flies different from humans. However, for lines of investigation intending to better understand the biology of GSCs, flies make an excellent model organism.
- Life cycle: Flies have a multi-stage life cycle involving pupae, whereas humans do not. There may be some key differences in germline regulation between these stages [Shcherbata et al, 2007].
- No freeze and store methods: There are no current methods to freeze and recover adult flies or larvae, so fly lines must be maintained continuously.
- Diapause: Fertility extension in flies is most readily achieved through diapause, a behavioral response to environmental inputs that is not immediately or intuitively applied to humans.
- Epigenetics: Drosophila contain extremely low DNA methylation levels compared to most other species, and even then the modifications bear only a loose similarity [Vidaurre and Chen, 2021]. It is believed that histone modification acts as the primary epigenetic control layer. This is a major difference between Drosophila and other models, with unknown consequences on reproductive aging.
Strains
Unlike other model organisms, there is no gold standard experimental fly strain, and lines between labs likely genetically differ greatly. Canton-S and w1118 strains are among those commonly used. There are no standard strains that have been commonly used for studying reproductive longevity. There exists, however, a vast variety of highly specialized and genetically modified lineages for nearly any type of study, stocked for immediate use. See key resources (especially Flybase) for additional information [Hales et al, 2015]
Basic Reproductive Biology
Fly Ovaries and the Stem Cell Niche
Drosophila continue to produce new oocytes even after they mature. Each fly has two ovaries consisting of 12 to 16 ovarioles. The germline stem cells (GSCs) in drosophila are regulated by cap cells and reside at the apical/anterior tip of the ovary, the germarium. From here, GSCs travel towards the cyst in the ovary’s posterior, developing through multiple intermediate stages (see below) before becoming a mature oocyte ready for laying. A detailed review of GSC development and fly ovaries can be found in Kirlly and Xie, 2007.
- Terminal Filament cells (TF): Most apical/anterior cells of ovariole which may express genes to regulate GSCs
- Cap Cells (CC/CpC) influence GSC behavior via direct cell-to-cell signaling and managing morphogen gradients.
- Germline Stem Cells (GSC) continually divide asymmetrically to produce a daughter GSC and a cystoblast.
- Cystoblasts (CB) are a differentiated form of GSCs. They divide asymmetrically into 16 cells, one of which will become an oocyte while the rest take on a support role.
- Escort Cells (EC) These cells remain stationary and act as structural elements of the ovary. They facilitate germline movement through the ovary.
Sex Determination
Though both X and Y chromosomes exist in Drosophila, they don’t have the same set of sex determination rules as in humans. Sexual differentiation is very complex, and determined not by number of X or Y chromosomes, but by a downstream regulatory mechanism that weighs the balance of X chromosomes to number of autosomal sets [Fox, 1956; Salz and Erickson, 2010]. See Gilbert’s paper (2000) for details. This may impact how genetic experiments with Drosophila can be translated to other species.
Sample Applications
Nutrition and Fertility
- Many key components of Drosophila metabolism are well-characterized, manipulable, and translatable to mammalian analogs. These pathways and their relationship to ovarian health is therefore somewhat conserved, making flies a great model for studying nutrition responses on a cellular level. The model is very useful for understanding fundamental mechanisms of the relationship between metabolism and fertility.
- Fly diet can be easily manipulated to mimic certain features of human diet choices. This can be used to robustly study the relationship between dietary composition and reproduction [Carvalho-Santos et al, 2020; Piper et al, 2013]. For example, high sucrose diets have been found to reduce fertility in flies. Mechanistically this appears to be caused by a reduced number of concurrent fully developed eggs in the ovary [Brookheart et al, 2017].
- A major question in reproductive aging is whether there is an inherent tradeoff between fertility and lifespan. Flies alter their rate of reproductive aging and development based on nutrition changes [Armstrong, 2020] and so may be used to study this phenomenon. Though such a tradeoff has been observed in flies and other organisms, preliminary experiments in flies have started to explore the idea that nutrition, lifespan and fecundity can be decoupled and how [Grandison et al, 2009].
- However, though certain nutrient response pathways are somewhat conserved between flies and humans, other metabolic systems may have limited translatability. For example, Drosophila fat storage occurs in the fat body, which is dissimilar to human fat storage and has different and additional physiological roles, such as detoxification and immune response [Armstrong, 2020; Musselman et al, 2013]. Since such macroscopic features of physiology are fundamentally tied with metabolism, some caution should be taken in interpreting translatability of certain metabolic data.
Energetics
- Experiments on ROS production and its effect on germline aging have been done on Drosophila [Easwaran et al, 2022]. ROS has been investigated in human oocyte aging and remains one of the leading theories for oocyte degradation over time [Goud et al, 2008]. It may be possible to use Drosophila models to gain insight into the role of ROS damage in human ovaries.
Germline development:
- Flies have been long used as a model to study how morphogen gradients drive development [Alberts et al, 2002]. This is because Drosophila are small and relatively simple organisms, but still have enough complexity to provide key developmental insights into human biology that other models like C elegans cannot. In the context of reproductive longevity, flies may be useful to determine the fundamental biological principles that guide differentiation of the germline and associated somatic cells. For example, flies have been used to identify JAK/STAT signaling gradients as responsible for germline cell niche differentiation [Shi et al, 2021]. Study of such processes in flies may be important for understanding the fundamental dynamics of ovarian reserve establishment
Oocyte Biology
- Similarly to other model organisms (i.e., C Elegans), Drosophila’s short generation time and relatively easy oocyte extraction methods can allow rapid and high throughput experimentation into oocyte development and dynamics. For example; see Greenblatt et al’s (2018) study of meiotic spindle dynamics in the fly.
Additional Reading
Key Reproductive Aging Studies Using Drosophila
There are a few key papers which first characterized the fertility decay and ovary aging in Drosophila melanogaster and pointed out potential cellular and molecular mechanisms underlying this process. In this section the main findings of these studies are summarized, plus an exploration of the strains and methodologies used which might explain certain differences in the results between studies. These differences should be taken in consideration when designing experiments studying reproductive aging and decay in Drosophila.
- David et al (1975) studied fecundity and fertility decay with age. In this study, the offspring of a cross between a strain of the vestigial mutant and Champetieres (a wild strain) was used. Females and males were housed in groups of 4 females and 5 males and the eggs were counted every day. There is no mention of whether males were changed to young males throughout the experiment, so the age of the male may also contribute to the reported results. The authors tested the effect of temperature in fertility decline and tested egg laying with females and males raised at 25 or 13ºC.
- A maximum of 80 eggs (25ºC) (or 40 eggs for females raised at 13ºC) was observed at day 4 and then gradually decreased but did not fully stop until day 50. The dynamics of egg laying seem to fit a sigmoid curve with females raised at 25ºC showing a decrease in the number of eggs laid by 2 eggs/day while females raised at 13ºC showed a decrease twice as slow - suggesting that their reproductive deterioration is slower.
- At 18ºC, the maximum number of eggs hatched peaks in the beginning of female’s life, after which it follows a nearly linear decrease. At 25ºC, the percentage of hatched eggs remains high and constant for 2 weeks, after which it decreases progressively, reaching values of around 20% in 35-40 day old females. There is also an increase in variability between females with age, a classical symptom of aging.
- Rauser et al (2006) analyzed the rate of fecundity decline using 10 independent populations of laboratory selected strains of D. melanogaster. Four females were housed with four males (12 day-old) and eggs were counted daily. It is not mentioned whether males were replaced during the course of the experiment.
- Under these conditions, fertility greatly slows at late ages, or plateaus at some small number of eggs laid per day slightly greater than zero. The viability of the eggs laid throughout life declines over all parental ages.
- Zhao and colleagues (2008) measured the average number of eggs produced by females per day since their eclosion. Two genotypes were analyzed, w1118 and Canton S. 3 females were housed with 3 males throughout the course of the experiment and males were replaced every 3 days by new 2-3-day old males to make sure females were always kept in a mated state. The authors characterize a number of features of fertility, including detailed analysis of cell number in the ovarioles.
- In both strains analyzed, the egg production had a peak at 3-4 days after eclosion and constantly decreased until it reached a minimum of near-zero eggs produced at around day 34 for w1118 and day 40 for Canton S. The percentage of unhatched eggs was also quantified. Data shows a decrease from 100% of hatching eggs to 40-35% in eggs from 40-day old females.
- The authors report a decrease in the number of germline stem cells from an average of 2.18 at day 3 post-eclosion to 1.66 in the ovarioles of 40 day old w1118 females, numbers which were similar in Canton S females. This ~25% decrease in the number of stem cells can not alone account for the dramatic decline in the number of eggs produced. The authors also reported that the proliferation of germline stem cells of 40-day old w1118 and Canton S females decreases by ~75%.
- The authors report a decrease in the number of germline stem cells from an average of 2.18 at day 3 post-eclosion to 1.66 in the ovarioles of 40 day old w1118 females, numbers which were similar in Canton S females. This ~25% decrease in the number of stem cells can not alone account for the dramatic decline in the number of eggs produced. The authors also reported that the proliferation of germline stem cells of 40-day old w1118 and Canton S females decreases by ~75%.
- Klepsatel at al (2013) followed the life histories of females from three recently caught, non-laboratory-adapted wild populations of D. melanogaster. In this paper, the authors use three recently collected outbred wild populations from Austria, South Africa and Zambia. One female was housed with two males and laid eggs were counted daily. Every 3 weeks, males were replaced with younger ones (1-14 day-old) to ensure results were not affected by failure of old males to successfully fertilize females. Hatched and unhatched eggs were counted after 48h at days 5, 15 and 20 and then daily thereafter.
- Populations varied in several components of reproductive life history, including ovariole number, various measures of age-specific fecundity, total fecundity, and egg production per ovariole, but did not differ in egg hatchability.
- Number of eggs per day in the three strains peaked in 3-10 day-old females and declined thereafter with increasing maternal age. Authors failed to confirm the existence of a late-life fecundity plateau (both for the number as well as hatchability of eggs) postulated in previous studies but found clear evidence for a pronounced fecundity peak.
- Females died approximately 6–10 days after having laid their last egg, suggesting that the onset of the post-ovipository period is a relatively good predictor of the time of death, but that this result was, on average, independent of how long individual females lived. Because the proportion of eggs that hatched declined to zero several days before egg production stopped, the authors redefined the length of the reproductive and post-reproductive period as follows: the period from eclosion until the day the last viable egg is laid represents the (true) reproductive period, whereas the period from the day the last viable egg is produced until death represents the (true) post-reproductive period. Using this definition they calculated the length of the reproductive period as being 20–22 days (approximately 60% of the total lifespan) and the post-reproductive lifespan as being 14–15 days, representing approximately 40% of the total lifespan. These data show that under optimal laboratory conditions, wild-caught fruit fly females can exhibit a very long post-reproductive phase.
- Since mature Drosophila oocytes are not as extensively stored in the ovaries under laboratory conditions as in the wild, Greenblatt et al (2019) developed a system to investigate how storage affects oocyte quality. Newly eclosed virgin female flies with immature ovaries were fed a nutrient-rich yeast paste that stimulates two young follicles per ovariole to develop to maturity. The withdrawal of yeast food after 24 hr prevents additional follicles from passing the nutritional checkpoint. In the absence of mating, the mature eggs are stored in the ovary indefinitely and not replaced.
- Greenblatt and colleagues measured the stability of stored oocytes over time by placing females with held, mature oocytes of known age with males, which stimulates the fertilization and deposition of these mature oocytes following mating. Oocytes stored for 0-5 days hatch at high rates (90–96%). However, oocytes stored for longer generate embryos with lower levels of hatching. At 29 ̊C, 25 ̊C or 20 ̊C, 50% of eggs fail to hatch after about 7, 12, or 23 days of storage, respectively.
- Stored mature oocytes showed no visible changes and retained the same protein content during 13 days of storage in the ovaries of protein-restricted females. Their eventual loss of developmental capacity was unaffected by maternal age.
- Bulk translation in 8 and 12-day-old oocytes was reduced to 59% and 43%, respectively, of the levels in 2 day oocytes. The data presented support the hypothesis that there is a decline in translation levels during oocyte aging.
- Among the genes showing a large reduction in translation were genes related to meiotic spindle organization and the spindle assembly checkpoint. Because of this, they investigated whether defects in the meiotic spindle could explain the reduced viability of aged oocytes. The meiotic spindles of 1 and 7-day old oocytes were usually bipolar and highly tapered. However, 13-day old oocytes showed abnormal spindles, such as unipolar, tripolar or fragmented. These defects are characteristic of mutants of meiotic spindle maintenance genes whose translation substantially declined in aged oocytes. The analysis of 1 and 14-day old oocytes found a striking correlation between the fraction of oocytes that hatch and the proportion of oocytes with bipolar spindles.
- To investigate whether errors of chromosome segregation are the major cause of reduced embryonic viability the level of embryonic development was analyzed for embryos derived from 1, 12 and 17-day old oocytes 4–8 hr after fertilization. Approximately half of embryos derived from oocytes stored for 12 days at 25 ̊C are able to hatch, but >95% fail to develop after 17 days. 100% of embryos derived from unstored oocytes progressed to stages 8–12. 58% of embryos from 12-day-old oocytes developed to stages 8–12, while the remaining 42% arrested during initial cleavage divisions of pre-blastoderm embryos. Almost 100% of embryos derived from 17-day-old oocytes remained arrested at pre-blastoderm stages.
IMPORTANT CONSIDERATION: Nutrition strongly affects fertility outcomes as well as egg laying behavior! For the sake of simplicity, the details of the diets used to rear the flies and for fertility assessment in the above-mentioned studies was not included in this summary. However, this factor should also be taken into consideration when comparing the results from the different studies as well as when planning an experimental design for studying reproductive aging and decay. Feeding females with different diets might impact fertility peaks, lifespan fertility, as well as rates of decay.
Features Modulating Reproductive Life History
Genetics
Putative genes indicated in the tradeoff between reproduction and lifespan, plus those that may influence fertility at later reproductive ages, are listed in Table 2 of Miller et al. Examples of well-studied genes in this list include Heatshock protein 70 (Hsp70) and Methuselah (mth). This report also references experiments (with the Indy gene) begun in flies that explore how fertility and longevity may be decoupled by targeting certain pathways. [Miller et al, 2014]
Nutrition
Fertility/Reproductive Output
Reduced nutrition can decrease fertility. The TOR, IGF, and Ecdysone pathways are implicated as the mechanisms for nutrition to influence fertility in drosophila [Ables et al, 2012]. High protein diets (60g vs 30g) can nearly double the number of eggs produced during peak egg laying period. [Watada et al, 2020]. Diet can also affect metabolic profiles in the offspring, which in turn may influence fertility [Buescher et al, 2013]. Other dietary nutrients including sugar, amino acids, vitamins and sterols have shown to be critical for animals to reach maximum fertility [Carvalho-Santos et al, 2020; Piper et al, 2013].
Sexual Maturity
Low nutrition can either trigger reproductive diapause or, independently, alter vitellogenesis. The process of yolk protein formation in the oocytes of non mammalian organisms, to reduce the rate of sexual maturation. Standard drosophila diets are composed of 1.5–10% w/v yeast, 5–10% cornmeal, 5–10% sugar, and 0.5–3% agar. Modifications can be made depending on the purpose of a particular experiment - typically ½ volume yeast for dietary restriction, 2-3x increase in sugar for high sugar diet, or the addition of coconut oil for high fat diet. [Armstrong, 2020l]. This simple modifiability allows for robust testing of dietary effects on reproductive function. For controlled and precise nutrient specific manipulations, chemically defined diets have been created and used to study the effect of dietary nutrients in longevity and fertility [Piper et al, 2013].
Integration of nutrient intake signals act via the IGF-1 pathway. Drosophila insulin receptor InR is an analog of c. elegans daf-2 - a highly-studied longevity gene that encodes the insulin receptor. Nutrient regulation has been connected to fertility and reproduction through InR. Altered nutrition intake triggers InR to invoke reproductive diapause via juvenile hormone [Tatar et al, 2001b]. In one study, mutation of the InR gene led to increased lifespan but sterility in females, while males were mostly sterile (20-35% of normal) [Tatar et al, 2001b] It is unclear whether TOR/FOXO is regulating ovarian function; though the mutation of numerous analogs in Drosophila strongly affect fertility, while others have no effect. [Giannakou and Partridge, 2007] Evidence suggests that nutrient intake acts independently of juvenile hormone for vitellogenesis [Richard et al, 2005]
Temperature
Sexual Maturity
Fly reproduction and life history can be dramatically altered by temperature. Early research found that raising Drosophila at 10C nearly doubled lifespan [Loeb and Northrup, 1916] Drosophila has a well characterized thermoreceptor system that feeds into gene expression [Barbagallo et al]. Correspondingly, altered temperature can modify longevity and reproduction through the induction of reproductive diapause (thought to act via juvenile hormone.) Flies kept at 11°C combined with altered photoperiod induce diapause and can maintain this arrest for up to 9 weeks with no significant change to mortality rates upon re-warming. However, fecundity drops by 70%, with severity of loss corresponding with duration of dormancy. [Tatar et al, 2001a] Flies kept at 10-12°C with altered photoperiods induced reproductive diapause [Saunders et al, 1989].
Photoperiod
The duration of daylight in a 24 hour window, referred to as photoperiod, affects the rate of sexual maturation. Large photoperiod alterations can induce reproductive diapause via juvenile hormone, delaying reproductive development [Tatar et al, 2001a]. The normal photoperiod setting for drosophila experiments is 12 hours light : 12 hours dark, often written as LD 12:12 [Schwartz et al, 1985; Saunders et al, 1989]. LD 10:14 and LD 16:8 photoperiods combined with 11°C temperatures induced reproductive diapause. [Tatar et a, 2001al] Diapause induction was also observed at LD 10:14 and below, as well as LD 18:6 and above when combined with 10-12°C temperatures [Saunders et al, 1989]. Altering photoperiod alone does not seem to significantly affect development time (~9% difference for low density, no difference for high density) [Giesel, 1988]. Photoperiod, in the context of reproductive aging, appears to be linked exclusively with reproductive diapause. Since this system is not present in humans, the applications seem limited to understanding the control of ovarian maturation in flies.
Density
High fly population density decreases fertility. The common explanation is that resource scarcity causes fertility reduction [Clark and Feldman, 1981]. Experiments on density showed the difference between 60 flies per vial vs. 2 flies per vial, demonstrating a reduction in egg production from ~8 to ~2 between low density and high density, accordingly. [Pearl, 1932]. Additionally, sex composition in housing can affect development; the presence of males accelerates ovarian development compared to singly-house females [Boake and Moore, 1996]. Density as an experimental tool has been phased out in favor of direct nutritional scarcity interventions as the mechanisms were discovered.
Mating and receptivity
Female receptivity peaks at >90% 1–2 days post-eclosion, then begins to decline approximately 7 days post-eclosion in virgin females or 5 days post-eclosion in mated females. By 3 weeks posteclosion, receptivity declines substantially to between 50% and 75% of maximal levels in previously virgin flies. Unmated females may live twice as long as mated females. [Miller et al, 2014]
Juvenile hormone
This hormone has particular importance because of its role as a node between reproductive function and aging - it is the mechanism that multiple different environmental effects funnel into. This single hormone appears to be critical for reproductive control in insects. Its analogs act as a throttle control for ovarian development in most other insects as well as flies. [Dubrovsky et al, 2002]. Low levels of juvenile hormone (JH) can induce reproductive diapause in flies [Tatar et al, 2001a], in fact, JH reduction alone is sufficient to increase lifespan at a cost of reduced fertility [Yamamoto et al, 2013]. Dosing flies with the juvenile hormone analog methoprene at .001ug/female induces oocyte maturation and reduced oxidative stress resistance even at low temperatures and altered photoperiods when flies would normally be in ovarian arrest. The maximum effect was observed at .1ug/female methoprene [Tatar et al, 2001b]. Flies were dosed with .05ug methoprene in acetone carrier, per fly, 3 hours after eclosion in another experiment to determine methoprene interaction with ecdysteroid production, which alters ovarian maturation [Schwartz et al, 1985].
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