Wings play an important role in dipteran life. Wing characteristics like size, coloration, and interference were found to be associated with male fitness. Many species in the genus Drosophila develop a male-specific wing pattern. These patterns appear as spots on the wings. In this work, we expose a set of recently collected out-bred lines of Drosophila biarmipes to a range of environmental stresses. The aim of this work was to find an association between wing pigmentation and stress response. We further looked the results with respect to the ‘good gene’ hypothesis. Males with higher wing-spot pigmentation did not associate with better stress handling. Interestingly, a majority of the traits showed negative or no trends where presence of a darker wing-spot could be associated to better handling of stressful environments. Contrary to this, desiccation tolerance and body size were found positively associated with darker wing-spot pigmentation. This work exposes the complexities behind secondary sexual characteristics and their association with male fitness and directs to relook the body size role in mate choice.  

Male wing spot pigmentation and tergite pigmentation

Egg collection was done at 23°C. To control egg density in the vials only 40 eggs in each tube were kept and the rest were removed before vials placed at a fixed temperature. Vials with eggs were moved to 23°C BOD incubator until eclosion. Upon eclosion males were separated and placed in fresh food vials. Males were aged for five days and preserved in ethanol. These males were then scored for wing-spot pigmentation. It was done using a stereozoom microscope (Jenco, USA). Ten males from each line were scored for wing spot and abdominal tergite pigmentation (David et al. 1990).

Female choice experiments

Virgin males and females were collected within 2-3 hours of eclosion. Males and females were kept in isolation (in groups of 4-5 individuals) for 5 days. Five-day-old adults were used in female choice experiments. With the help of a mouth aspirator flies were transferred to mating chambers (diameter: 1.5 cm, height: 0.5 cm). Each mating chamber was labeled to identify both the line and the wing spot status of the male. Mating assays were done between 8-11 am. Two male individuals (each from a different line) differing in wing-spot pigmentation were added to each mating chamber already having a female from other lines separated by a sliding wall. The wall within the mating chamber are then pulled off and the camera was turned on. The videos were taken using Samsung 65X intelli-zoom recorder. The behavioural recordings were done for one hour duration. To access successful mating pairs numbers, videos were examined later. A total of 20 pairs were studied.

Starvation and desiccation tolerance assays

Freshly eclosed males were collected in groups of ten in fresh food vials. Males were kept in these vials for four-five days before subjecting them for starvation and desiccation tolerance assays. For both starvation and desiccation tolerance assays, the starting number of the adults in the tubes was ten.

 For starvation, the 4-5 day old males were moved to vials containing 3mL solution of 2% agar. The starvation tolerance was also measured at 23°C. The dead flies were counted every 3 hours till mortality reached 50% and thereafter every hour. Desiccation assay was performed in regular narrow Drosophila culture vials. Dry conditions were maintained using Silica gel (Sigma-Aldrich, USA), along with a foam plug to separate the flies from the silica gel. Vials were then sealed using parafilm. Silica gel adsorbs the moisture from the vials and maintains humidity less than 2%. The tubes were kept at 23°C. The mortality was measured at an interval of 3 hours until half of the flies were dead and thereafter each hour.

Heat-knockdown assay

Ten males from each line were collected and aged for five days in isolation before subjecting them for heat-knockdown time measurment. Each fly was transferred to a 4mL small glass tube and an airtight lid was placed over it. No CO2 was used during this entire process.  These tubes were arranged on a custom build plexiglass rack (to hold the vials when submerged in a water bath). The entire rack was submerged into a glass chamber full of water where water temperature was maintained at 39°C. This glass chamber was connected to a waterbath (Equitron Medica, India). As fly knock-down time was recorded. The experiment performer did this manually. To avoid any sort of bias labels were randomized so that experiment performer did not know the actual identity of the tubes.  

Body size measurement

Males were aged for five days before measuring thorax length. To measure thorax length males were placed on a glass slide under a dissecting microscope and a digital image was captured at a set focal length. Images were processed in ImageJ software using a calibration slide. For body size measurements five males from each line were used and the thorax sizes were collected in mm.

Fecundity Assay

For fecundity assay eggs were collected from each line in the groups of 30-40 eggs. All the procedures were done at 23°C. Within two hours of eclosion virgin flies were collected and aged for five days. Three lines with darker spots and three lines with lighter spots were chosen for this assay. Five males from each of these lines were collected in isolation.  Three intermediate wing-spot pigmentation lines were selected for the female collection. Ten females were collected from each line. Half of the females from each line were allowed to mate with darker wing-spot male lines and the rest half were allowed to mate with lighter wing-spot male lines. Pairs were kept in tubes for the next 24 hours at 23°C. After 24 hours males were removed from each vial (with the help of a mouth aspirator) and the female were transferred to fresh tubes. Following this transfer, vials were examined for daily eggs laid. This was done after transferring females to fresh tubes. Egg counts were recorded for fifteen days.