a. Ancestral populations:
The two ancestral populations used in this study are called LH and LHst, both large laboratory adapted populations of Drosophila melanogaster. The LH population was established by Lawrence Harshman from 400 gravid wild caught females. This population is maintained at an effective population size >5000[45]. LHst was derived by introgression of a benign autosomal ‘scarlet eye’ marker to the LH genetic background and is maintained at an Ne>2500. The LH and LHst populations are genetically equivalent except for one locus which has no effect on their fitness. The additive genetic variation in the LHst population is maintained through periodic back cross with LH[46]. Both populations are maintained at standard laboratory condition (temperature = 25oC, relative humidity ≈ 60%) in a 12:12 dark: light cycle and are reared on corn-meal molasses food. Detailed population maintenance is described in[47]. Briefly, in a given generation, 2-3-day-old adult flies from rearing vials (95 mm height × 25 mm diameter) are mixed and redistributed into fresh food vials -16 males and 16 females in each - containing a limiting quantity of dried yeast granules. The flies are kept there for two days after which they are allowed to oviposit for 18 hours in fresh vials with food. These vials are controlled for density (~150 eggs /vial) and incubated to start the next generation.
b. Selection regimes:
The selection regimes are derived from LHst. Initially three populations, C1-3, were derived and maintained for 5 generations. The maintenance of the C populations differed from that of LHst in that adult males and females were collected as virgins and held in same-sex vials with 8 individuals/vial and combined in 1:1 sex ratio (16 males and 16 females) once they were 2-3 days old with measured amount of live yeast paste instead of granules. Thereafter the maintenance protocol is the same as that of LHst. After 5 generations, two more selection regimes, F1-3 and M1-3, were derived from each of the C populations where operational sex ratios where biased towards males and females respectively. In these populations, 2-3 day-old virgin adults were combined in their respective sex ratios, i.e., Male: Female ~ 1:3 and 3:1 for F and M respectively. Note that the populations sharing the same subscript share a common ancestry and are handled simultaneously, independent of those having a different subscript. Thus, each subscript constitutes a “statistical block”. Details of maintenance and selection history is described in [10].
c. Stndardization:
Nongenetic parental effects [48]can lead to misinterpretation of multi-generation selection experiment results. To equalize such effects across selection regimes, all selected populations were passed through one generation of standardization where selection was removed, i.e., they were maintained in ancestral conditions [49]. Adult progeny produced by this generation were used for the experiment.
d. Bacterial culture
We used three pathogens for this study: gram negative bacteria Providencia rettgeri[50], gram negative bacteria Pseudomonas entomophila L48 [51], and gram positive bacteria Staphylococcus succinus subsp. Succinus, strain PK-1 (Ss) [52]. All three bacteria are natural isolates obtained from wild caught Drosophila. For making the bacterial suspension for infections, bacterial culture was grown at 27°C (Pe) and 37°C (Ss and Pr) till OD = 1.0 ± 0.1 from a glycerol stock maintained at -80oC. Following this, cells were pellet down and suspended in equal volume of 10 mM MgSO4 before infection. For Pr, the suspension was concentrated to OD 2.0 ± 0.1 before infection.
e. Infection protocol:
Flies were put under light CO2 anaesthesia and infected by pricking with a needle (Minutein pin 0.1 mm, Fine Science Tools, CA) dipped in bacterial suspension (bacteria suspended in 10 mM MgSO4) in the thorax (Gupta et al.2013). To control for injury, a separate set of flies were pricked with a tungsten needle dipped in sterile 10 mM MgSO4 (sham).
f. Experimental Treatments:
For each of the three separate assays (using the three pathogens), the following experimental protocol was used:
Experimental males were collected within 6 hours of eclosion from pupae, which ensured their virginity, since in these populations it takes the flies ~8 hours to attain sexual maturity. These males were kept in vials provided with corn-meal molasses food at a density of 8 males /vial. On 12th day post egg collection (i.e., 2-3-day-old adult) flies from each selection regime were randomly assigned to two groups: ‘virgin’ and ‘mated’.
In the ‘virgin’ treatment, virgin males were transferred to vials containing fresh food as they were. In the ‘mated’ treatment, males from each vial were combined with virgin LHst females (8 / vial). A total of 15 vials were set up per treatment per selection regime per block. Ten (n = 80) and 5 (n= 40) vials were used for infection and sham (control) respectively. All pricking was done on 14th day post egg collection and were transferred to vials containing fresh food following infection. Males in the ‘mated’ treatment were separated from females while anaesthetized for pricking and were maintained in single sex vials.
g. Mating and courtship measurements:
We measured the number of mating obtained by selection regime males in the ‘mated’ treatment in the assay where Pr was used. All the vials in the ‘mated’ treatment were observed manually. A total of 22 observations were taken over a period of 48 hours, with more frequent observations during the light-dark and dark-light transition (+/- 2 hours before and after the transitions). Number of mating pairs and courting males were recorded at each observation timepoint. Average number of mating and courting male per vial were calculated and used as the unit of analyses using the following formula: (see Equation 1 in the Supplementary Files)
We used this as a proxy of the total amount of mating acquired or courtship displayed by a male over the period of 48 hours.
h. Measure of infection response:
For Pe and Ss, response to pathogenic infection was measured in terms of survivorship post infection by observing vials for mortality every three hours post infection for ~100 hours post infection. For Pr, since mortality was low (<5%) and did not differ from the sham control, response was measured as the ability of the host to clear bacteria using a previously established method [23]. Briefly, 20 hours post infection, 6 flies from each vial were sampled randomly and divided into groups of three. They were then crushed using a mortar inside micro-centrifuge tubes containing 100 μL MgSO4 and plated on LB-Agar plates using an automated spiral plater (WASP spiral plater, Don Whitley Scientific, UK). Three replicate plates were plated from each group of three flies. After growing the bacteria in their respective optimum temperatures, CFUs were counted using a plate reader (Acolyte colony counter, Don Whitley Scientific, UK). Average CFUs per fly obtained from each group was used as unit of analysis.
i. Statistical Analyses:
All analyses were performed in R. Survivorship (for Pe and Ss) was analyzed using Cox’s Proportional hazards model. Time to death was recorded for each fly and flies not dead till the last time were treated as censored data. For each of the pathogens, data were modelled either using block as a random factor or excluding Block using R package “Coxme”[53]using the following two expressions: (see Expressions 1 and 2 in the Supplementary Files)
Since analysis of deviance revealed no effect of block(analysis of deviance test: χ22 = 0.72, p = 0.69 for Pe; χ22 = 0.01, p = 0.99 for Ss), data from all three blocks were pooled and the cumulative data were then tested for difference in survivorship. We compared the Cox partial likelihood (log-likelihood) estimates across treatments and selection regimes.
In the case of Pr, colony count data was natural log transformed and normality was verified using a Shapiro – Wilk test. To test for various factors data were then subjected to the following glm models using package “lme4” [54] (all relevant R codes are provided as SI):
Effect of selection regime and mating status (mated vs virgin) on CFU: (see Expression 3 in the Supplementary Files)
Effect of selection regime on mating and courtship: (see Expression 4 in the Supplementary Files)
Test for the effect of courtship on CFU in mated males belonging to the two selection regimes: (see Expression 5 in the Supplementary Files)