Fly husbandry
Two kinds of laboratory Drosophila melanogaster populations were used in this study. The Control (C) populations were on 21 days egg to egg discrete generation cycle, while the Selected (S) populations were derived from the controls by direct selection for faster pre-adult development and indirect selection for extended longevity. Detailed protocols adopted in rearing and maintenance of C and S populations are explained previously13. Briefly, each of the three C populations were cultured in 40 vials with 6 ml standard banana-jaggery media (SM) at a density of 40-50 eggs per vial and incubated at SLC for full 12 days13. At the end of 12 days, all emerging flies from the 40 vials were transferred to pre-labeled plexi-glass population cages and provided with fresh food every alternate day till day 18. On day 18, fresh food plate was supplemented with live yeast-acetic acid paste. Eggs for initiating the next generation were collected on day 21 from the previous egg collection day. Each of the three S populations was derived from the three C populations by collecting 160 vials of 60-80 eggs per 6 mL banana-jaggery food vial. The vials were incubated at SLC. The early emerging 15-20 flies (as ascertained by empty pupal cases) from each vial were transferred to pre-labeled population cages. Two sister cages were maintained per S population to avoid adult crowding. The S population cages too were provided with fresh food plates every alternate day till 50% mortality was noticed in any of the cages, at which point all cages were provided fresh food plates supplemented with live yeast-acetic acid paste for three days following which eggs for starting next generation were collected. The eggs obtained from the two sister cages of a given population were mixed and redistributed into 160 vials.
Originally, the Control populations (also called as JB populations) were derived from IV populations51 and are described in detail in Prasad et al.17. The selected and control populations had been through 134 and 242 generations respectively at the time of being used in this study. To remove non-genetic parental effects which might appear due to the differences in maintenance regime, both the S and C populations were run through common rearing conditions for 1 generation at a moderate density of 50 eggs per 6 mL banana-jaggery media vial and 40 vials per population before being used in this study. The egg collection from the S and C populations were staggered by their developmental time difference to synchronize the emergence of adults. All adults emerging from each of the 40 vials of a given population were transferred to pre-labeled population cage with fresh SM plate. These populations are referred to as standardized flies13, 17. Embryos for all the experiments were obtained from these standardized flies (SF).
Generation of Critical size adults and Normal-sized adults
Synchronized eggs were collected from SF, evenly spread on agar-agar plates and incubated at SLC. Freshly hatched larvae (~22 h post-egg-laying) were harvested using a fine camel hair brush and transferred to Petri-plates (5.5 mm diameter, Tarson) with 2000 µL of LSM (Liquid Standard Media) at a density of 30 larvae per plate13. Twenty such plates per population were incubated at SLC. The larvae from all the 20 plates were re-harvested from LSM plates after 64 and 72 h (post-egg-lay) for S and C populations respectively, washed with RO (Reverse osmosis) water, rolled on tissue towel and randomly transferred (25 larvae per vial) to vials containing 6 mL non-nutritive agar-agar or SM and incubated at SLC13. At every 6 hour interval, the emerging flies from these vials were collected, sorted according to their gender and held as virgins in pre-labeled holding vials with 6mL SM till use in further assays. The adults that emerged from vials containing non-nutritive agar are referred to as critical-sized (CS) adults, while those that emerged from vials containing SM are referred to as normal-sized (NS) adults.
Macromolecule quantification during larval life
Glycogen and lipid content were estimated using Van Handel’s method52 and the protein content was estimated using Smith’s method53 with minor modifications.
All three macromolecules were quantified for pre-critical stages (L2 to L3 transition stage- 48 h for both C and S populations; Early L3 stage-56 h and 64 h for S and C populations respectively), critical size stage (64 h and 72 h for S and C) and post-critical stages (Late L3 stages - 72 h and 104 h; before pupation L3- 80 h and 112 h for S and C populations respectively) of larval life. All the sampling time intervals mentioned are from the time of transfer of newly hatched larvae to LSM plates. Assays are presumed to have been performed using synchronized larvae from standardized flies in both control and selected populations. The detailed protocols used in the estimation of the macromolecules are as follows:
-
Glycogen estimation
Five randomly chosen larvae were homogenized in 400 µL of 2% Na2SO4. 80 µL of homogenate was aliquoted into 5 mL Eppendorf tube, to which 184 µL of Na2SO4 and 3736 µL of the (fresh) mixture of chloroform and methanol (1:1) was added. The tubes with the mix were centrifuged (Eppendorf, 5430R) at 14000 r.p.m. for 10 minutes at 4 °C. The supernatant was discarded and the pellet was air-dried for 10 minutes. The pellet was resuspended in 2000 µL Anthrone reagent and heated at 90 °C in water-bath for 10 min. Aliquots were kept on ice for 5 min following which absorbance was measured at 625 nm on ELISA plate reader (ECIL micro scan, MS5605A). The assay was repeated in triplicate per population and means of the triplicate measures were used in statistical analysis.
-
Protein estimation
Precipitation assay was done before quantification of protein followed by BCA method of protein quantification53. Five randomly chosen larvae were homogenized in 400 µL of 2% Na2SO4. Then 80 µL of homogenate was aliquoted and 500 µL of 0.15% Deoxycholate was added to the aliquot. After the incubation period of 10 min on ice, 1000 µl 3M Trichloroacetic acid (TCA) was added. The aliquots were centrifuged at 8500 r.p.m. (Eppendorf, 5430R) for 15 min at 4 °C. Protein was precipitated at the base of each aliquot. Pellets were washed with HCl and air-dried. BCA reagent was added to each of the pellets, resuspended and heated in water-bath at 60 °C for 10 min. Absorbance was recorded at 562 nm using ELISA plate reader (ECIL micro scan, MS5605A). The assay was repeated in triplicate per population and means of the triplicates were used in statistical analysis.
-
Lipid estimation
Five randomly chosen larvae were homogenized in 200 µL of PBS (1X). 1 mL of freshly prepared methanol and chloroform mixture (1:1) was added to the homogenate and vortexed. This was followed by centrifugation at 4 °C at 4000 r.p.m. (Eppendorf, 5430R). Two phases of the solution were separated. From the lower layer of solution, 200 µL of the solution was taken in an aliquot and was evaporated completely at 90 °C, using water-bath. 50 µL of H2SO4 was added post evaporation and incubated at 60 °C for 10 min, cooled on ice for 5-7min., following which 1000 µL of Vanillin reagent was added and incubated at room temperature for 30 min. Absorbance was taken at 525 nm using ELISA plate reader (ECIL micro scan, MS5605A). The assay was repeated in triplicates per population and means of triplicate were used in statistical analysis.
All the biomolecules converted to their energy equivalents54 and compared.
Adult life-history traits
-
Copulation latency and copulation duration
Copulation latency (the time lag between the time of emergence and initiation of copulation) and copulation duration (the time difference between initiation and termination of copulation) were ascertained by pairing freshly emerged flies Zero-day flies with 3-day old mature flies of the opposite gender
-
Life-time oviposition and longevity
One day old virgins from holding vials were used in this assay. The flies were anaesthetized using CO2 and a female and male pair were transferred to fresh vials with 3 mL SM. A total of 20 pairs per treatment were set up. Any fly that did not wake up within 1 h of transfer was replaced with a new fly of the same gender. Further, any fly that died within the first 24 h of set up was also replaced by a fresh fly. The pair of flies were transferred to fresh 3 mL SM vials every 24 hours. The eggs laid in the preceding 24 h were counted under stereo zoom microscope (Carl Zeiss Binocular stereozoom microscope, Stemi 305) and recorded. Census records were also maintained till the death of all flies. The average life-time oviposition and life-span were estimated from this primary data (Fig. 2a).
Data analysis
In all cases except survival probability function, univariate analysis of variance, under general linear model (GLM) using SPSS v. 22 was carried out and population means were used as units of analysis with selection, larval growth stage and fly type as fixed variables and replication as a random variable55-57. Hence, only fixed-factor effects and interactions could be tested for significance13, 17. The significance of adult survival probability curves was analyzed using Kaplan-Meier log-rank test58.