Changes in Body Melanisation and Not Body Size Affect Mating Success in Drosophila immigrans 29
Materials and Methods
Field Collections Copulating pairs and non-
copulating flies of D. immigrans from decay-
ing fallen fruits in a highland locality (winter of
2007–2008) were aspirated. Flies were collected
in the morning (6:00–8:00 a.m.) when they were
actively involved in mating. The copulating pairs
were collected in food vials with aspirator. After
90 min, the unsuccessful flies (male and female
both) that is, the one that did not copulate were
aspirated in separate food vials (noncopulating
flies).
D. immigrans ( n = 120–180) were also col-
lected from three altitudinal sites (a low-, mid-
and highland locality) covering a range of 761–
2708 m in October–November (2007–2008)
using banana bait traps and net sweeping. The
sampling sites were characterized by their lati-
tude, altitude, average annual temperature (Tave)
and relative humidity (RH) on the basis of cli-
matological tables obtained from Indian Institute
of Tropical Meteorology (IITM; http://www.tropmet.
res.in). For each population, 45 isofemale lines
were set up. Wild populations are adapted to dif-
ferent climatic conditions, but between popula-
tion differences can be analysed on the basis of
common garden experiments. Thus, flies of each
population were reared in biological oxygen de-
mand (BOD.) incubators at 21 ± 0.2 °C. The den-
sity was controlled by limited egg laying period
(6–8 h) on cornmeal–yeast–agar medium (70–80
eggs/vial, 37 × 100 mm).
Culture Conditions For each population, 2–3
replicates of isofemale lines were maintained
for simultaneous analysis of body melanisation,
wing length and mating related traits. All experi-
ments were initiated soon after collections and
performed with successive generations. The ex-
perimental conditions were made uniform with
temperature controlled room set at 21 °C. Fur-
ther, for each season, G 1 and G 2 (generations 1
and 2) of the wild caught flies were analysed to
avoid possible effects of laboratory adaptations
or inbreeding effects.
Trait Measurements Lines were selected on the
basis of body size (wing length) and body mela-
nisation, i.e. (a) lines similar in body size but dif-
fering in body melanisation (b) lines similar in
body melanisation but varying in body size from
the 45 isofemale lines prepared. Ten randomly
chosen individuals per isofemale line were in-
vestigated. Body size (wing length) and body
melanisation were simultaneously measured in
wild caught and laboratory reared individuals.
For each fly, wing length (in millimetre) was
measured from the thorax articulation to the tip
of third longitudinal vein under Olympus SZ-11
microscope fitted with a micrometer.
Melanisation was estimated from dorsal as
well as lateral views of the abdomen giving val-
ues ranging from 0 (no pigment) to 10 (complete
darkness) for each of the six visible abdominal
tergites. For visual scoring of melanisation under
stereo zoom microscope (Olympus, http://www.olym-
pus.com) the melanisation score (0–10) for each
tergite was multiplied with its relative size. Since
the abdominal tergites (2nd to 7th) differ in size,
the total area of each tergite was transformed into
its relative size, i.e. 0.66, 0.88, 1.0, 0.88, 0.66
and 0.33 for 2nd to 6th tergites, respectively, for
both the sexes. However, the size of 7th tergite
differed between sexes, i.e. 0.22 for males and
0.33 for females. The abdomen of each fly minus
viscera was mounted on a slide and total body
melanisation per fly was also estimated through
Biowizard image analysis software (Dewinter
Optical Inc., http://www.dewinterindia.com). All ex-
periments were done on assorted dark and light
flies (melanisation difference ~ 20–25 %) and as-
sorted small and large flies (wing length differ-
ence ~ 0.50 mm) of isofemale lines from a high-
land population. Laboratory selection experiment
for body melanisation was carried out in a mid-
land population (Solan) at 21 °C for 18 genera-
tions and different traits were analysed.Analyses of Mating-Related Traits For mat-
ing-related traits (ML, CD, ovariole number
and fecundity), assorted isofemale lines were
analysed. Mating experiments were conducted
when flies were more active: early in the morn-
ing (6:00–8:00 a.m.). During experiments, two