Materials and Methods for Drosophila melanogaster Study

This document provides information on the materials and methods used in a study on Drosophila melanogaster. It includes details on the strains used, rearing conditions, and crosses performed. The study aimed to investigate sex-linked inheritance in fruit flies.

Materials and Methods
Two strains of Drosophila melanogaster were used in the study, with the females having the
cinnabar phenotype, and males being white-eyed. Cinnabar eyes are considered a recessive
characteristic of sex-linked inheritance in fruit flies. Both the species for the parental generation
were obtained from stock center of Drosophila species, at the university. The primary reason for
selecting fruit flies for this experiment can be accredited to the fact that they have a short life
span, can be easily crossed, and are feasible for conducting chromosome analysis (Yamamoto et
al.). The genotypes for the two parents were as follows:
1. XcXc (cinnabar females) * XwY (white-eyed males)
2. XcY (cinnabar males) * XwXw(white-eyed females)
Both the species were reared on a diet that comprised of molasses medium and standard
cornmeal. The medium had supplementary live yeast grains, with the aim of promoting egg
laying. The two parental species were maintained in 25-30ml vials that were closed with foam
plugs at a temperature of around 22-23 °C.
Two crosses were performed for the experiment. The first one involved crossing between the
parental phenotypes of 4 cinnabar males and 2 white-eyed females. The second one involved a
reciprocal cross between parental phenotypes of 4 cinnabar females and 2 white-eyed males. The
female sexed pupae for the two different species (white-eyed and cinnabar), were placed in
different vials. The virgin enclosed female flies were moved to new vials every day, followed by
aging them, before conducting the cross. The crossing was performed by adding male flies in the
jars, once the females were deemed virgin. The process involved a ratio of 2:1. The fact was
taken into consideration that males were able to mate in an efficient manner, if they had attained
maturity before three days, or more, Mating process occurred rapidly and the females were found

to lay their eggs, immediately after mating. The adults were removed from the jar, once the
presence of adequate number of larvae was established. Removal of the adult flies from the two
separate jars, one for each type of cross, was conducted 7 days after the cross occurred. This
helped in easy distinguishing between the F1 generation and the parents. Following three weeks
after crossing between parental white females and cinnabar males, the F1 generation offspring
were all wild-type (red eye color). On making the reciprocal cross, wild type females and
cinnabar males were obtained.
After obtaining the separate F1 generation from the two crosses conducted between the
parent flies, 8 cinnabar males were again made to cross with 8 white apricot females. The F1
generation was kept in same vials where the parents had been placed. After removal of the parent
flies, yeast was added to the vials, and they were closed using cotton and foam plugs. After
taking out the parental generation, the larvae for F1 were located in the white mush. The flies
were raised at a temperature of 22°C.There were few dead parents located at the mush bottom.
The third and final instar were observed on the fifth day. By the end of week 1, the larvae began
the roaming stage, and pupariation commenced 120 hours, after laying the eggs. At the end of
two weeks, the F1 adults began emerging from the case of the pupa.
There was no F2 generation for the cross between all wild-type flies that were obtained
after mating between 4 cinnabar males and 2 white females. On the other hand, the cross
conducted between 8 cinnabar males and 8 white apricot females of F1 generation resulted in 4
dissimilar phenotypes in the F2 generation namely, (i) white apricot, (ii) wild-type, (iii) cinnabar,
and (iv) orange (double mutant).
Chi square test was run to observe the differences between the phenotypes of the flies obtained at
the F2 generation. The rationale behind using this test was that it allows exploring the association

between categorical variables. Prominence of the differences between all the flies obtained in the
F2 generation were determined by using the formula given below:
where, fe denoted the expected frequency if there was no association between the variables, and
fo denoted the observed frequency (the observed number of males and females for each
phenotype). In order to draw a conclusion about the proposed hypothesis with 95% confidence
interval, p value lesser than 0.05 was considered significant for the results (Sharpe). Upon
obtaining F2 generation scores with p<0.05, it would be concluded that the variables were not
independent of each other, and there existed a statistical correlation between the phenotypes.
Results of the P generation cross (F1 generation)
When D. Melanogaster was crossed with 4 cinnabar males and 2 white females, the F1 offspring
of the F1 generation were wild type. However, when cinnabar females were crossed with white
apricot males, the F1 generation offspring produced wild-type females (red) and cinnabar males.
 Cinnabar males * White females = Wild type
 Cinnabar females * White apricot males: Wild type females and cinnabar males
The main unexpected development during the P generation cross is unexpected death of the
adult after one week in both the sexes. In the crosses, after mating was done between the parental
generation, the parents were removed (P generation) and the offspring were kept inside the jar
separately in order to ensure easy counting and analysis of the eye colour of the offspring. In the
separate jars, where the adults (P generation) and the offspring were kept (F1) generation, yeast
was added. According to Kinjo, Hirotoshi, Kunimi, and Nakai, female fruit fly lay eggs that

hatch into maggots. The maggots feed on the liquid year solution present at the bottom of jar. At
around 77 degree F (temperature condition optimum for the growth of the Drosophila), it takes
nearly one week for the fruit flies to develop under the three maggot stages and then pupate and
produce a second generation of adult files. Though the adults both male and females were kept
under the same environmental condition, there are unexpected deaths among the parental adults
and there were no specific pattern of death across the gender and the death was random between
both the sexes (both male and female).
Evaluation of Data
The cross for the F1 generation showed wild-type offspring however, the cross of the F2
generation failed to produce any distinct results. In F2 generation, 8 cinnabar males (XCY) were
crossed with white apricot females (XwXw). It was expected the F2 generation will produce wild-
type eye colour females and males will be cinnabar along with double mutant (orange).
However, the observed offspring failed to match with the predicted rate. However, the results
showed double mutant (orange), white apricot, wild type and cinnabar. The evaluation of the
data was done separately. The evaluation of the data highlighted that the observed results (150)
of the orange or double mutant were close to the expected value (96.75). All the calculations
were done separately.
The results of the F1 generation
Phenotype # observed (both
sexes)
#
expected
(observed - expected)^2 /
expected
White apricot
Males=8
Females=36
44 96.75 28.76
Wildtype
Males=98
227 290 13.68

Females=129
Orange (Double
Mutant)
Males=78
Females=72
150 96.75 29.308
Cinnabar
Males=40
Females=55
95 32.25 122.09
Total/Sum 516 516 193.838
Table 1: Results of the F1 generation cross: F2 generation
The table here shows the observed phenotypes in the F2 generation of a cross of D.
melanogaster, cinnabar females and white apricot males. The expected phenotypes, of both
sexes, are shown based on the hypothesis that the F2 generation females would be wild-type eye
color, males would be cinnabar and there would be a double mutant. The observed offspring
does not fit the predicted ratio of white apricot: 3/16, wild-type: 9/16, orange (double mutant):
1/16 and cinnabar: 3/16 (X2 = 193.838, p value= <0.0001;d.f.= 3)
Phenotype of the double mutant
The phenotype of the double mutant in the F2 generation cross highlighted orange color.
This is a new eye color that is not observed among the parents. Deep orange eye color is defined
as classic eye color genes in Drosophila. The deep orange eye color is a part of protein complex
located in the endosomal compartments (Lőrincz, Péter, et al). The reason for the generation of
the unexpected cross results can be explained through the “Batson-Dobzhansky Muller” model in
the domain of the rise of the genetic incompatibilities in the hybrids. For example, the genotype
of AABB (ancestral population) over time gets divided into two isolated populations. This leads
to the generation of new mutation in one population while in the second population the B allele

becomes mutated to b allele. Here a and be alleles are mutually incompatible. However, this is
not an issue in pure species population, as these two alleles do not come in direct contact.
However, when the individuals of these populations are mated they produce hybrid. This can be
regarded as incompatibility and has a potential to negatively impact on the fitness of the hybrid.
In the figure highlight below, the black arrows indicate the process of divergence and the double-
headed green-headed black arrows is the symbol of the incompatibility.
Figure: Graphical representation of “Batson-Dobzhansky-Mulle” model
(Source: Li, Wang, and Zhang)
Reason for unexpected results
However, taking into consideration of the Batson-Dobzhansky-Muller‟ model, still the
generation of the four different phenotypes is something unnatural in the F2 generation. The
generation of the four different phenotypes is not supported through both the crosses that were
set-up for the experiment. Both the crosses yielded chi-squares values, which were above the
range of 0.5 range in relation to the degree of freedom. In the F2 generation, the double mutant
resulted from the cross were X-linked females and it demonstrated orange eye colour. This

resulted in the dis-approval of the null hypothesis with the prescribed ration of the Mendelian F2
generation of 9:3:3:1 as the P-value was more than 0.5 and failed to satisfy the expected ratio.
The ratio of the cross in the second generation was 2.38: 1.57 : 0.46 : 1 (wild type :
double mutant : white apricot : cinnabar). Thus the ratio highlights a strong divergence from the
ratio of the second generation Mendelian di-hybrid cross (9: 3: 3: 1).
The error was in the F1 generation cross that has occurred between the cinnabar females
and white apricot males. The cross between the cinnabar females (XcXc) and white apricot
males (XwY) showed wild type females and cinnabar males. The reason behind this is cinnabar
gene and white apricot gene is X-linked recessive (Dickinson, William and David Sullivan). This
means that in order to express the colored eye in females, both the X-chromosome of the female
genotype need to have the mutant allele. However, in male, having the single mutant allele will
express the phenotype as male has only one X chromosome, so cross between Cinnabar females
(XcXc) and White apricot males (XwY) will yield double mutant females (XcXw) and cinnabar
males (XcY). There is no scope to get wild-type females as female gets one X-chromosome from
her father (in this case it would be Xw) and another X chromosome from her mother (Xc).
However, the results showed wild type female. The phenotypic expression of male was however,
correct as it showed cinnabar males. Justification is male gets one X-chromosome from his
mother only and here the mother contains cinnabar (Xc) allele.
In another cross in the F1 generation with the parental genes cinnabar males (XcY) were
crossed with white females (XwXw). White apricot gene is X-linked recessive (Dickinson,
William and David Sullivan). However, the results showed wild type males and females. If the
cross was proper then the results would have been XcXw (double mutant female), XwY (white
eyed male). There is no chance to get a wild type phenotype for both the sexes male receive one

x chromosome from his mother and in this case the both the X chromosome of female is has w
allele and thus it is not feasible to have a wild type male.

References
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