DOI: 10.5061/dryad.08kprr551
title: Male wing spot morphology and physiological responses do not coincide with the good-gene hypothesis

description: 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.  
description: <em><strong>Male wing
 spot pigmentation and tergite
 pigmentation</strong></em>
 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).
 <em><strong>Female
 choice experiments</strong></em>
 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.
 <em><strong>Starvation and desiccation tolerance assays
 </strong></em> 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
 <em>Drosophila </em>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.
 <em><strong>Heat-knockdown assay
 </strong></em> 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.  
 <em><strong>Body size
 measurement</strong></em>
 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. <em><strong>Fecundity Assay
 </strong></em> 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.