Cross of white eyed males and cinnabar females of Drosophila melanogaster

This study explores the cross between white eyed males and cinnabar females of Drosophila melanogaster, resulting in multiple strains of eye pigmentation. The hypothesis, materials and methods, and statistical analysis are discussed.

Running head: BIOLOGY
Biology
Name of the Student
Name of the University
Author Note

1
BIOLOGY
Title: Cross of white eyed males and cinnabar females of Drosophila melanogaster
yielding in multiple strains of eye pigmentation.
H1: While crossing cinnabar maleand white apricot female drosophila melanogaster,
females of the F2 generation will express wild-type eye color, and the males would be
cinnabar. Location of the white apricot gene on the X-chromosome would also lead to double
mutants (orange color) in the F2 generation.
H0: While crossing cinnabar maleand white apricot female drosophila melanogaster,
females of the F2 generation will not express wild-type eye color, and the males would not be
cinnabar. Although the white apricot gene is located on the X-chromosome, no double
mutants (orange color) will be obtained in the F2 generation.
Materials and Methods
Drosophila stock and fly maintenance- 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

2
BIOLOGY
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. The table given below shows the proportion
of molasses medium that were provided to the fruit flies:
Agar 8g
Cold water 200ml
Boiling water 1000ml
Yeast 15g
Propionic acid 5ml
Molasses 55ml
Cornmeal 80g
Table 1- Components of vial media
The stir bar was placed inside a beaker having 2 liters capacity, and cold water, agar,
yeast, and cornmeal were mixed into it. The beaker was placed on a hotplate, followed by
turning heat to high levels, which put the bar into motion. This prevented deposition of the
food at the bottom, and also ensured that it did not get burnt. Boiling water was added to the
medium. Following boiling of the mixture for at least 30-40 seconds, molasses was added.
The molasses was thoroughly mixed, prior to removal of the food from heat (Tatar, Post and
Yu). Upon reaching a temperature of 60o C, propionic acid was added and stirred, till it got
evenly mixed with the medium. This food was then poured into vials, while ensuring that
they contained at least 1cm of food, and were refrigerated for future use. Efforts were taken
to ensure that the vials were rehydrated, since it acted as the major source of water for the
Drosophila adults and larvae.
Prior to adding the flies to the media, it was taken out of the refrigerator and warmed
to room temperature. Steps were taken to ensure that the media filled the vials till 1/5th or
2/5th full. It was kept overnight for curing, with a cloth used to cover the vials. This kept flies

3
BIOLOGY
from laying eggs inside. Yeast and plugs were added the next day, and unused media were
refrigerated.
Environment- Although Drosophila can be easily raised at room temperature, the
optimum raising condition is near a temperature of 25ºC. 60% humidity is considered best for
rearing the flies, which helps in reducing the generation time (Lin et al.). It took around 9
days for an egg to transform into an adult under this condition.
Anesthetizing- The flies were anesthetized using carbon dioxide. It worked by
immobilizing the flies for a long duration, and did not have any adverse effects. CO2 mats, a
bottle that acted as CO2 source, and a delivery system was required for the procedure.
Crossing flies-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

4
BIOLOGY
females and cinnabar males, the F1 generation offspring were all wild-type (red eye colour).
On making the reciprocal cross, wild type females and cinnabar males were obtained.
F1 cross- 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, eclosion started and 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).
Statistical analysis- 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:

5
BIOLOGY
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
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
Unexpected developments
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

6
BIOLOGY
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 44 96.75 28.76

7
BIOLOGY
Males=8
Females=36
Wildtype
Males=98
Females=129
227 290 13.68
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

8
BIOLOGY
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)

9
BIOLOGY
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

10
BIOLOGY
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.

11
BIOLOGY
References
Dickinson, William J., and David T. Sullivan. Gene-enzyme systems in Drosophila. Vol. 6.
Springer Science & Business Media, 2013.
Kinjo, Hirotoshi, Yasuhisa Kunimi, and Madoka Nakai. "Effects of temperature on the
reproduction and development of Drosophila suzukii (Diptera: Drosophilidae)." Applied
entomology and zoology 49.2 (2014): 297-304.
Li, Chuan, Zhi Wang, and Jianzhi Zhang. "Toward Genome-Wide Identification of Bateson–
Dobzhansky–Muller Incompatibilities in Yeast: A Simulation Study." Genome biology and
evolution 5.7 (2013): 1261-1272.
Lin, Suewei, et al. "Neural correlates of water reward in thirsty Drosophila." Nature
neuroscience 17.11 (2014): 1536.
Lőrincz, Péter, et al. "iFly: The eye of the fruit fly as a model to study autophagy and related
trafficking pathways." Experimental eye research 144 (2016): 90-98.
Sharpe, Donald. "Your chi-square test is statistically significant: Now what?." Practical
Assessment, Research & Evaluation 20 (2015).
Tatar, Marc, Stephanie Post, and Kweon Yu. "Nutrient control of Drosophila
longevity." Trends in Endocrinology & Metabolism 25.10 (2014): 509-517.
Yamamoto, Shinya, et al. "A Drosophila genetic resource of mutants to study mechanisms
underlying human genetic diseases." Cell 159.1 (2014): 200-214.

12
BIOLOGY

13
BIOLOGY